Single domain antibodies directed against stat3

ABSTRACT

This invention provides compositions and methods to treat a condition or disease without the use of exogenous targeting sequences or chemical compositions. The present invention relates to single-domain antibodies (sdAbs), proteins and polypeptides comprising the sdAbs that are directed against intracellular components that cause a condition or disease. The invention also includes nucleic acids encoding the sdAbs, proteins and polypeptides, and compositions comprising the sdAbs. The invention includes the use of the compositions, sdAbs, and nucleic acids encoding the sdAbs for prophylactic, therapeutic or diagnostic purposes.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication No. 62/067,908, filed on Oct. 23, 2014, U.S. ProvisionalPatent Application No. 62/148,656, filed on Apr. 16, 2015, U.S.Provisional Patent Application No. 62/188,353 filed on Jul. 2, 2015, andU.S. Provisional Patent Application No. 62/210,795, filed on Aug. 27,2015, the contents of which are incorporated herein by reference intheir entirety.

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file titled“Sequence_Listing_STP25.txt,” created Sep. 30, 2015, last modified Oct.22, 2015, and is 83,000 bytes in size. The information in the electronicformat of the Sequence Listing is incorporated herein by reference inits entirety.

BACKGROUND

The use of single-domain antibodies (sdAbs) as single antigen-bindingproteins or as an antigen-binding domain in larger protein orpolypeptide offers a number of significant advantages over the use ofconventional antibodies or antibody fragments. The advantages of sdAbsinclude: only a single domain is required to bind an antigen with highaffinity and with high selectivity; sdAbs can be expressed from a singlegene and require no post-translational modification; sdAbs are highlystable to heat, pH, proteases and other denaturing agents or conditions;sdAbs are inexpensive to prepare; and sdAbs can access targets andepitopes not accessible to conventional antibodies.

There are a number of diseases or conditions, such as cancer, that arecaused by aberrant intracellular or transmembrane components such asnucleotides and proteins. Elimination of the aberrant components can beused to prevent or treat the diseases or conditions. There are a numberof pharmacological compounds available for treatment, but the compoundscan be ineffective, undeliverable, or toxic to unaffected cells.

Other treatments include the use of therapeutic proteins or agents thatcontain an exogenous targeting sequence so that the therapeutic agentcan be recognized by receptors in the cell membrane, enabling thetherapeutic agent to cross the cell membrane and enter the cell. Oncethe therapeutic agent is inside the cell, the therapeutic agent caninteract with the target component in order to treat the disease.However, the use of exogenous targeting sequence can limit the cell typethat is targeted by the therapeutic agent, and adds to the cost ofmanufacturing the therapeutic agent.

For the foregoing reasons, there is a need for compositions and methodsto treat or prevent a disease that do not rely on exogenous targetingsequences or chemical compositions in order to enter the cell, and thatare effective in targeting only the affected cells in the body.

The present invention relates to single-domain antibodies (sdAbs),proteins and polypeptides comprising the sdAbs. The sdAbs are directedagainst intracellular components that cause a condition or disease. Theinvention also includes nucleic acids encoding the sdAbs, proteins andpolypeptides, and compositions comprising the sdAbs. The inventionincludes the use of the compositions, sdAbs, proteins or polypeptidesfor prophylactic, therapeutic or diagnostic purposes. The invention alsoincludes the use of monoclonal antibodies directed towards the sdAbs ofthe invention.

SUMMARY

One embodiment of the invention is a single-domain antibody (sdAb)directed against an intracellular component. The intracellular componentcan be, for example, a protein, nucleic acid, lipid, carbohydrate,STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b, STATE, TNF-alpha, and KRAS.

In another embodiment, the invention is directed towards an anti-STAT3sdAb. Optionally, the anti-STAT3 sdAb comprises an amino acid sequenceset forth in SEQ ID NO:3 or SEQ ID NO:4.

In another embodiment, the invention is directed towards an isolatedpolypeptide comprising an amino acid sequence encoding an anti-STATsdAb, such as, for example, the polypeptide set forth in SEQ ID NO:3 orSEQ ID NO:4.

In yet another embodiment, the invention is directed towards a hostcell, and the host cell expresses the amino acid sequence of the sdAbsuch as, for example, the amino acid set forth in SEQ ID NO:3 or SEQ IDNO:4.

One embodiment of the invention is a pharmaceutical compositioncomprising a sdAb, or a polypeptide, and a pharmaceutically acceptablecarrier. Optionally, the sdAb comprises an anti-STAT3 sdAb comprising anamino acid sequence set forth in SEQ ID NO:3 or SEQ ID NO:4, and thepolypeptide comprises an isolated polypeptide comprising an amino acidsequence set forth in SEQ ID NO:3 or SEQ ID NO:4.

Another embodiment of the invention is a method to diagnose a disordermediated by STAT3 in a subject, the method comprising the steps of a)contacting a biological sample with the sdAb, or a polypeptide; b)determining the amount of STAT3 in the biological sample; and c)comparing the amount determined in step (b) with a standard, adifference in amount indicating the presence of the disorder.

Another embodiment of the invention is a method of preventing ortreating a disease or disorder, or preventing recurrence of a diseasemediated by STAT3, or for use in the treatment of cancer, or diseasescaused by abnormal cellular proliferation, comprising administering ananti-STAT3 sdAb, or a polypeptide, to a subject in need thereof.Optionally, the sdAb comprises an anti-STAT3 sdAb comprising an aminoacid sequence set forth in SEQ ID NO:3 or SEQ ID NO:4 and thepolypeptide comprises an isolated polypeptide comprising an amino acidsequence set forth in SEQ ID NO:3 or SEQ ID NO:4.

One embodiment of the invention is an anti-TNF-alpha sdAb. Optionally,the anti-TNF-alpha sdAb comprises an amino acid sequence set forth inSEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:7. The invention also comprises anisolated polypeptide comprising an amino acid sequence set forth in SEQID NO:5, SEQ ID NO:6 or SEQ ID NO:7.

Another embodiment of the invention is a host cell expressing the aminoacid sequence set forth in SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:7.

In another embodiment, the invention is also a pharmaceuticalcomposition comprising a sdAb or a polypeptide and a pharmaceuticallyacceptable carrier. Optionally, the sdAb comprises an anti-TNF-alphasdAb comprising an amino acid sequence set forth in SEQ ID NO:5, SEQ IDNO:6 or SEQ ID NO:7 and the polypeptide comprises an isolatedpolypeptide comprising an amino acid sequence set forth in SEQ ID NO:5,SEQ ID NO:6 or SEQ ID NO:7.

Another embodiment of the invention is a method to diagnose a disordermediated by TNF-alpha in a subject, the method comprising the steps ofa) contacting a biological sample with a sdAb or a polypeptide; b)determining the amount of TNF-alpha in the biological sample; and c)comparing the amount determined in step (b) with a standard, adifference in amount indicating the presence of the disorder.

In one embodiment, the invention describes a method of preventing ortreating a disease or disorder or recurrence of a disease or disordermediated by TNF-alpha, or for use in the treatment of cancer, ordiseases caused by abnormal cellular proliferation, comprisingadministering an anti-TNF-alpha sdAb, or a polypeptide, to a mammal inneed thereof. Optionally, the anti-TNF-alpha sdAb comprises an aminoacid sequence set forth in SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:7 andthe polypeptide comprises isolated polypeptide, the isolated polypeptidecomprising an amino acid sequence set forth in SEQ ID NO:5, SEQ ID NO:6or SEQ ID NO:7.

One embodiment of the invention is an anti-KRAS sdAb. Optionally, theanti-KRAS sdAb comprises an amino acid sequence set forth in SEQ IDNO:2. In one aspect, the invention comprises an isolated polypeptide,wherein the isolated polypeptide comprises an amino acid sequence setforth in SEQ ID NO:2. In another aspect, the invention comprises a hostcell expressing the amino acid sequence set forth in SEQ ID NO:2.

Another embodiment of the invention is a pharmaceutical composition,comprising a sdAb or a polypeptide, and a pharmaceutically acceptablecarrier. Optionally, the sdAb comprises an anti-KRAS sdAb comprising anamino acid sequence set forth in SEQ ID NO:2 and the polypeptidecomprises isolated polypeptide comprising an amino acid sequence setforth in SEQ ID NO:2.

An additional embodiment of the invention is method to diagnose adisorder mediated by KRAS in a subject, the method comprising the stepsof a) contacting a biological sample with a sdAb or a polypeptide; b)determining the amount of KRAS in said biological sample; and c)comparing the amount determined in step (b) with a standard, adifference in amount indicating the presence of the disorder.Optionally, the sdAb comprises an anti-KRAS sdAb comprising an aminoacid sequence set forth in SEQ ID NO: 2 and the polypeptide comprisesisolated polypeptide comprising an amino acid sequence set forth in SEQID NO:2.

The invention also comprises a method of treating a disease using ananti-KRAS sdAb, the method comprising administering an effective amountof an anti-KRAS sdAb to a subject in need thereof.

In one embodiment, the invention describes a method of preventing ortreating a disease or disorder, or the recurrence of a disease ordisorder, mediated by KRAS, or for use in the treatment of cancer, ordiseases caused by abnormal cellular proliferation, comprisingadministering an anti-KRAS sdAb or a polypeptide, to a mammal in needthereof. Optionally, the anti-KRAS sdAb comprises an amino acid sequenceset forth in SEQ ID NO: 2 and the polypeptide comprises isolatedpolypeptide comprising an amino acid sequence set forth in SEQ ID NO: 2.

In one embodiment, the invention describes a method of administering thesdAb of the invention, the method comprising intravenous administration,intramuscular administration, oral administration, rectaladministration, intraocular administration, enteral administration,parenteral administration, subcutaneous administration, transdermaladministration, administered as eye drops, administered as nasal spray,administered by inhalation or nebulization, topical administration, andadministered as an implantable drug.

In another embodiment, the invention describes a method of treating adisease, preventing a disease or preventing the reoccurrence of adisease using the sdAb of the invention in combination with one or morecompounds. Optionally, the one or more compounds is a transcriptionalinhibitor.

In another embodiment, the invention describes a method of measuring thelevels of a sdAb, the method comprising the steps of a) generating amouse monoclonal antibody directed against one or more domains of thesdAb; b) performing an immunoassay to determine the amount of sdAb in asubject; and c) quantifying the amount of sdAb in the subject.

DRAWINGS

These and other features, aspects, and advantages of the presentinvention will become better understood with regard to the followingdescription, appended claims, and accompanying drawings where:

FIG. 1 is a schematic map of VHH13 anti-STAT3 sdAb expression vectorpTT21-stt VHH13;

FIG. 2 is a schematic map of VHH14 anti-STAT3 sdAb expression vectorpTT21-stt VHH14;

FIG. 3 depicts the results of an immunoprecipitation assay usinganti-STAT3 bacterial VHH13 STAT3 (SEQ ID NO:3) and anti-STAT3 bacterialVHH14 STAT3 (SEQ ID NO:4);

FIG. 4 depicts the results of an immunoprecipitation assay usinganti-STAT3 bacterial VHH13 STAT3 (SEQ ID NO:3);

FIG. 5 illustrates the growth inhibition of anti-STAT3 bacterial VHH13(SEQ ID NO:3) sdAb in the MDA-MB-231 xenograft model, dosed at 0.5mg/kg/day;

FIG. 6 illustrates the growth inhibition of anti-STAT3 bacterial VHH13(SEQ. ID. NO. 3) sdAb in MDA-MB-231 xenograft model at doses rangingfrom 1 mg/kg twice daily to 2 mg/kg twice daily or 2 mg/kg/day;

FIG. 7 illustrates the growth inhibition of anti-STAT3 bacterial VHH13(SEQ ID NO:3) sdAb in the MDA-MB-231 xenograft model, dosed at 5mg/kg/twice daily;

FIG. 8 illustrates the growth inhibition of anti-STAT3 bacterial VHH13(SEQ ID NO:3) sdAb in the DU145 xenograft model, dosed at 5 mg/kg/twicedaily;

FIG. 9 illustrates the growth inhibition of anti-STAT3 bacterial VHH13(SEQ ID NO:3) sdAb in the PANC-1 xenograft model, dosed at 5 mg/kg/twicedaily;

FIG. 10 illustrates the growth inhibition of anti-STAT3 bacterial VHH13(SEQ ID NO:3) sdAb in the MCF-7 xenograft model, dosed at 1 mg/kg/threetimes daily;

FIG. 11 illustrates the growth inhibition of anti-STAT3 bacterial VHH13(SEQ ID NO:3) sdAb in the BT-474 xenograft model, dosed at 1 mg/kg/threetimes daily;

FIG. 12 illustrates the cytotoxicity of TNF-alpha in U937 cells;

FIG. 13 illustrates the cytotoxicity of Staurosporine in U937 cells; and

FIG. 14 illustrates inhibition of TNF-alpha cytotoxicity byanti-TNF-alpha sdAbs.

DESCRIPTION

As used herein, the following terms and variations thereof have themeanings given below, unless a different meaning is clearly intended bythe context in which such term is used.

The terms “a,” “an,” and “the” and similar referents used herein are tobe construed to cover both the singular and the plural unless theirusage in context indicates otherwise.

The term “antigenic determinant” refers to the epitope on the antigenrecognized by the antigen-binding molecule (such as an sdAb orpolypeptide of the invention) and more in particular by theantigen-binding site of the antigen-binding molecule. The terms“antigenic determinant” and “epitope” may also be used interchangeably.An amino acid sequence that can bind to, that has affinity for and/orthat has specificity for a specific antigenic determinant, epitope,antigen or protein is said to be “against” or “directed against” theantigenic determinant, epitope, antigen or protein.

As used herein, the term “comprise” and variations of the term, such as“comprising” and “comprises,” are not intended to exclude otheradditives, components, integers or steps.

It is contemplated that the sdAbs, polypeptides and proteins describedherein can contain so-called “conservative” amino acid substitutions,which can generally be described as amino acid substitutions in which anamino acid residue is replaced with another amino acid residue ofsimilar chemical structure and which has little or essentially noinfluence on the function, activity or other biological properties ofthe polypeptide. Conservative amino acid substitutions are well known inthe art. Conservative substitutions are substitutions in which one aminoacid within the following groups (a)-(e) is substituted by another aminoacid within the same group: (a) small aliphatic, nonpolar or slightlypolar residues: Ala, Ser, Thr, Pro and Gly; (b) polar, negativelycharged residues and their (uncharged) amides: Asp, Asn, Glu and Gln;(c) polar, positively charged residues: His, Arg and Lys; (d) largealiphatic, nonpolar residues: Met, Leu, Ile, Val and Cys; and (e)aromatic residues: Phe, Tyr and Trp. Other conservative substitutionsinclude: Ala into Gly or into Ser; Arg into Lys; Asn into Gln or intoHis; Asp into Glu; Cys into Ser; Gln into Asn; Glu into Asp; Gly intoAla or into Pro; His into Asn or into Gln; Ile into Leu or into Val; Leuinto Ile or into Val; Lys into Arg, into Gln or into Glu; Met into Leu,into Tyr or into Ile; Phe into Met, into Leu or into Tyr; Ser into Thr;Thr into Ser; Trp into Tyr; Tyr into Trp; and/or Phe into Val, into Ileor into Leu.

A “domain” as used herein generally refers to a globular region of anantibody chain, and in particular to a globular region of a heavy chainantibody, or to a polypeptide that essentially consists of such aglobular region.

The amino acid sequence and structure of an sdAb is typically made up offour framework regions or “FRs,” which are referred to as “Frameworkregion 1” or “FR1”; as “Framework region 2” or“FR2”; as “Frameworkregion 3” or “FR3”; and as “Framework region 4” or “FR4,” respectively.The framework regions are interrupted by three complementaritydetermining regions or “CDRs,” which are referred as “ComplementarityDetermining Region 1” or “CDR1”; as “Complementarity Determining Region2” or “CDR2”; and as “Complementarity Determining Region 3” or “CDR3,”respectively.

As used herein, the term “humanized sdAb” means an sdAb that has had oneor more amino acid residues in the amino acid sequence of the naturallyoccurring VHH sequence replaced by one or more of the amino acidresidues that occur at the corresponding position in a VH domain from aconventional 4-chain antibody from a human. This can be performed bymethods that are well known in the art. For example, the FRs of thesdAbs can be replaced by human variable FRs.

As used herein, an “isolated” nucleic acid or amino acid has beenseparated from at least one other component with which it is usuallyassociated, such as its source or medium, another nucleic acid, anotherprotein/polypeptide, another biological component or macromolecule orcontaminant, impurity or minor component.

The term “mammal” is defined as an individual belonging to the classMammalia and includes, without limitation, humans, domestic and farmanimals, and zoo, sports, and pet animals, such as cows, horses, sheep,dogs and cats.

As used herein, “pharmaceutically acceptable carrier” is intended toinclude any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration. Suitable carriersare described in the most recent edition of Remington's PharmaceuticalSciences, a standard reference text in the field. Preferred examples ofsuch carriers or diluents include, but are not limited to, water,saline, Ringer's solutions, dextrose solution, PBS (phosphate-bufferedsaline), and 5% human serum albumin. Liposomes, cationic lipids andnon-aqueous vehicles such as fixed oils may also be used. The use ofsuch media and agents for pharmaceutically active substances is wellknown in the art. Except insofar as any conventional media or agent isincompatible with a therapeutic agent as defined above, use thereof inthe composition of the present invention is contemplated.

A “quantitative immunoassay” refers to any means of measuring an amountof antigen present in a sample by using an antibody. Methods forperforming quantitative immunoassays include, but are not limited to,enzyme-linked immunosorbent assay (ELISA), specific analyte labeling andrecapture assay (SALRA), liquid chromatography, mass spectrometry,fluorescence-activated cell sorting, and the like.

The term “solution” refers to a composition comprising a solvent and asolute, and includes true solutions and suspensions. Examples ofsolutions include a solid, liquid or gas dissolved in a liquid andparticulates or micelles suspended in a liquid.

The term “specificity” refers to the number of different types ofantigens or antigenic determinants to which a particular antigen-bindingmolecule or antigen-binding protein molecule can bind. The specificityof an antigen-binding protein can be determined based on affinity and/oravidity. The affinity, represented by the equilibrium constant for thedissociation of an antigen with an antigen-binding protein (KD), is ameasure for the binding strength between an antigenic determinant and anantigen-binding site on the antigen-binding protein: the lesser thevalue of the KD, the stronger the binding strength between an antigenicdeterminant and the antigen-binding molecule (alternatively, theaffinity can also be expressed as the affinity constant (KA), which is1/KD). As will be clear to one of skill in the art, affinity can bedetermined depending on the specific antigen of interest. Avidity is themeasure of the strength of binding between an antigen-binding moleculeand the antigen. Avidity is related to both the affinity between anantigenic determinant and its antigen binding site on theantigen-binding molecule and the number of pertinent binding sitespresent on the antigen-binding molecule. Specific binding of anantigen-binding protein to an antigen or antigenic determinant can bedetermined by any known manner, such as, for example, Scatchard analysisand/or competitive binding assays, such as radioimmunoassays (RIA),enzyme immunoassays (EIA) and sandwich competition assays.

As used herein, the term “recombinant” refers to the use of geneticengineering methods (for example, cloning, and amplification) used toproduce the sdAbs of the invention.

A “single domain antibody,” “sdAb” or “VHH” can be generally defined asa polypeptide or protein comprising an amino acid sequence that iscomprised of four framework regions interrupted by three complementaritydetermining regions. This is represented asFR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. An sdAb of the invention also includes apolypeptide or protein that comprises the sdAb amino acid sequence.Typically, sdAbs are produced in camelids such as llamas, but can alsobe synthetically generated using techniques that are well known in theart. As used herein, the variable domains present in naturally occurringheavy chain antibodies will also be referred to as “VHH domains,” inorder to distinguish them from the heavy chain variable domains that arepresent in conventional 4-chain antibodies, referred to as “VH domains,”and from the light chain variable domains that are present inconventional 4-chain antibodies, referred to as “VL domains.” “VHH” and“sdAb” are used interchangeably herein. The numbering of the amino acidresidues of a sdAb or polypeptide is according to the general numberingfor VH domains given by Kabat et al. (“Sequence of proteins ofimmunological interest,” US Public Health Services, NIH Bethesda, Md.,Publication No. 91). According to this numbering, FR1 of a sdAbcomprises the amino acid residues at positions 1-30, CDR1 of a sdAbcomprises the amino acid residues at positions 31-36, FR2 of a sdAbcomprises the amino acids at positions 36-49, CDR2 of a sdAb comprisesthe amino acid residues at positions 50-65, FR3 of a sdAb comprises theamino acid residues at positions 66-94, CDR3 of a sdAb comprises theamino acid residues at positions 95-102, and FR4 of a sdAb comprises theamino acid residues at positions 103-113.

The term “synthetic” refers to production by in vitro chemical orenzymatic synthesis.

The term “target” as used herein refers to any component, antigen, ormoiety that is recognized by the sdAb. The term “intracellular target”refers to any component, antigen, or moiety present inside a cell. A“transmembrane target” is a component, antigen, or moiety that islocated within the cell membrane. An “extracellular target” refers to acomponent, antigen, or moiety that is located outside of the cell.

A “therapeutic composition” as used herein means a substance that isintended to have a therapeutic effect such as pharmaceuticalcompositions, genetic materials, biologics, and other substances.Genetic materials include substances intended to have a direct orindirect genetic therapeutic effect such as genetic vectors, geneticregulator elements, genetic structural elements, DNA, RNA and the like.Biologics include substances that are living matter or derived fromliving matter intended to have a therapeutic effect.

As used herein, the phrases “therapeutically effective amount” and“prophylactically effective amount” refer to an amount that provides atherapeutic benefit in the treatment, prevention, or management of adisease or an overt symptom of the disease. The therapeuticallyeffective amount may treat a disease or condition, a symptom of disease,or a predisposition toward a disease, with the purpose to cure, heal,alleviate, relieve, alter, remedy, ameliorate, improve, or affect thedisease, the symptoms of disease, or the predisposition toward disease.The specific amount that is therapeutically effective can be readilydetermined by an ordinary medical practitioner, and may vary dependingon factors known in the art, such as, e.g., the type of disease, thepatient's history and age, the stage of disease, and the administrationof other therapeutic agents.

The present invention relates to single-domain antibodies (sdAbs) thatare directed against intracellular components, as well as to proteinsand polypeptides comprising the sdAbs and nucleotides encoding theproteins and polypeptides. The invention can also relate to sdAbs thatare directed against intercellular, transcellular and extracellulartargets or antigens. The invention also includes nucleic acids encodingthe sdAbs, proteins and polypeptides, and compositions comprising thesdAbs. The invention includes the use of the compositions, sdAbs,proteins or polypeptides for prophylactic, therapeutic or diagnosticpurposes.

SdAbs have a number of unique structural characteristics and functionalproperties which make sdAbs highly advantageous for use as functionalantigen-binding domains or proteins. SdAbs functionally bind to anantigen in the absence of a light chain variable domain, and canfunction as a single, relatively small, functional antigen-bindingstructural unit, domain or protein. This distinguishes sdAbs from thedomains of conventional antibodies, which by themselves do not functionas an antigen-binding protein or domain, but need to be combined withconventional antibody fragments such as Fab fragments or ScFv's fragmentin order to bind an antigen.

SdAbs can be obtained using methods that are well known in the art. Forexample, one method for obtaining sdAbs includes (a) immunizing aCamelid with one or more antigens, (b) isolating peripheral lymphocytesfrom the immunized Camelid, obtaining the total RNA and synthesizing thecorresponding cDNAs, (c) constructing a library of cDNA fragmentsencoding VHH domains, (d) transcribing the VHH domain-encoding cDNAsobtained in step (c) to mRNA using PCR, converting the mRNA to ribosomedisplay format, and selecting the VHH domain by ribosome display, and(e) expressing the VHH domain in a suitable vector and, optionallypurifying the expressed VHH domain.

Another method of obtaining the sdAbs of the invention is by preparing anucleic acid encoding an sdAb using techniques for nucleic acidsynthesis, followed by expression of the nucleic acid in vivo or invitro. Additionally, the sdAb, polypeptides and proteins of theinvention can be prepared using synthetic or semi-synthetic techniquesfor preparing proteins, polypeptides or other amino acid sequences.

The sdAbs of the invention will generally bind to all naturallyoccurring or synthetic analogs, variants, mutants, alleles, parts andfragments of the target, or at least to those analogs, variants,mutants, alleles, parts and fragments of the target that contain one ormore antigenic determinants or epitopes that are essentially the same asthe antigenic determinant or epitope to which the sdAbs of the inventionbind in the wild-type target. The sdAbs of the invention may bind tosuch analogs, variants, mutants, alleles, parts and fragments with anaffinity and/or specificity that is the same as, or that is higher thanor lower than the affinity and specificity with which the sdAbs of theinvention bind to the wild-type target. It is also contemplated withinthe scope of the invention that the sdAbs of the invention bind to someanalogs, variants, mutants, alleles, parts and fragments of the targetbut not to others. In addition, the sdAb of the invention may behumanized, and may be monovalent or multivalent, and/or multispecific.Additionally, the sdAbs of the invention can bind to the phosphorylatedform of the target protein as well as the unphosphorylated form of thetarget protein. sdAbs can be linked to other molecules such as albuminor other macromolecules.

In addition, it is within the scope of the invention that the sdAbs aremultivalent, that is, the sdAb can have two or more proteins orpolypeptides which are directed against two or more different ofepitopes of the target. In such a multivalent sdAb, the protein orpolypeptide may be directed, for example, against the same epitopes,substantially equivalent epitopes, or different epitopes. The differentepitopes may be located on the same target, or it could be on two ormore different targets.

It is also contemplated that the sequence of one or more sdAbs of theinvention may be connected or joined with one or more linker sequences.The linker can be, for example, a protein sequence containing acombination of serines, glycines and alanines.

It is also within the scope of the invention to use parts, fragments,analogs, mutants, variants, alleles and/or derivatives of the sdAbs ofthe invention, as long as these are suitable for the described uses.

Since the sdAbs of the invention are mainly intended for therapeuticand/or diagnostic use, they are directed against mammalian, preferablyhuman, targets. However, it is possible that the sdAbs described hereinare cross-reactive with targets from other species, for example withtargets from one or more other species of primates or other animals (forexample, mouse, rat, rabbit, pig or dog), and in particular in animalmodels for diseases and disorders associated with the disease associatedwith the targets.

In another aspect, the invention relates to a nucleic acid that encodesan sdAb of the invention. Such a nucleic acid may be, for example, inthe form of a genetic construct.

In another aspect, the invention relates to host or host cell thatexpresses or is capable of expressing an sdAb of the invention, and/orthat contains a nucleic acid encoding a sdAb of the invention. Sequencesof the sdAbs can be used to insert into the genome of any organism tocreate a genetically modified organism (GMO). Examples include, but arenot limited to, plants, bacteria, viruses, and animals.

The invention further relates to methods for preparing or generating thesdAbs, nucleic acids encoding the sdAbs, host cells expressing orcapable of expressing such sdAbs, products and compositions containingthe sdAbs of the invention.

The invention further relates to applications and uses of the sdAb, thenucleic acids encoding the sdAbs, host cells, products and compositionsdescribed herein. Such a product or composition may, for example, be apharmaceutical composition for treatment or prevention of a disease, ora product or composition for diagnostic use. sdAbs can be used in avariety of assays, for example ELISA assays and mass spectrometry assaysto measure the serum and tissue levels of the sdAbs.

In another aspect, a nucleic acid encoding one or more sdAb of theinvention can be inserted into the genome of an organism to treat orprevent diseases.

The present invention generally relates to sdAbs, as well as to proteinsor polypeptides comprising or essentially consisting of one or more ofsuch sdAbs, that can be used for prophylactic, therapeutic and/ordiagnostic purposes.

The methods and compositions detailed in the present invention can beused to treat disease described herein, and can be used with any dosageand/or formulation described herein or otherwise known, as well as withany route of administration described herein or otherwise known to oneof skill in the art.

The sdAbs of the invention, in particular the anti-STAT3 VHH, theanti-KRAS VHH, and the anti-TNF-alpha VHH of the present invention, canbe used for treatment and prevention of malignant diseases including,but not limited to: multiple myeloma, leukemias (HTLV-1 dependent,erythroleukemia, acute myelogenous leukemia (AML), chronic myelogenousleukemia (CML), and large granular lymphocyte leukemia (LGL), lymphomas(EBV-related/Burkitt's, mycosis fungoides, cutaneous T-cell lymphoma,non-Hodgkins lymphoma (NHL), anaplastic large-cell lymphoma (ALCL),breast cancers, triple-negative breast cancers, head and neck cancers,melanoma, ovarian cancers, lung cancers, pancreatic cancers, prostatecancers, sarcomas, osteosarcoma, Kaposi's sarcoma, Ewing's sarcoma,hepatocellular cancers, glioma, neuroblastoma, astrocytoma, colorectalcancers, Wilm's tumors, renal cancers, bladder cancers, endometrialcancers, cervical cancers, esophageal cancers, cutaneous squamous cellcancers, basal cell cancers, and any metastatic cancers. The sdAbs canbe used in cancer patients to help prevent or reduce weight loss orcachexia due to cancer.

The sdAb, in particular the anti-STAT3 and the anti-TNF-alpha sdAbs ofthe present invention, can also be used for treatment and prevention ofdiseases such as, but not limited to: autoimmune diseases (e.g.,rheumatoid arthritis, ulcerative colitis, Crohn's disease, bacterialinduced colitis, asthma, scleroderma, lupus, encephalomyelitis,arteritis, vasculitis, glomerulonephritis, uveitis, uveoretinitis,multiple sclerosis), polycystic kidney disease, dermatologic diseases(e.g., psoriasis, alopecia areata, atopic dermatitis,keloids/hypertrophic scars, lipoma, Padget's disease, and actinickeratosis), Hidradenitis suppurativa, transplantation (e.g., solidorgan, bone marrow, hand, face, limbs, any body part), musculardystrophy and muscle wasting associated with cancers and aging,endometriosis, macular degeneration, retinal degeneration, stroke,epilepsy, traumatic brain and spinal cord injuries, hypertension,cardiac hypertrophy, Alzheimer's disease, pulmonary artery hypertension,type 2 diabetes mellitus, and ankylosing spondylitis. Additionally sdAbscan target orphan diseases. Examples of these rare orphan diseasesinclude, but are not limited to, triple negative breast cancers,pancreatic cancers, AML (acute myeloid leukemia), head and neck cancers,multiple myeloma, and chemo-resistant cancers.

Viral infections can be treated by targeting intracellular viralproteins in infected cells. Viral proteins, such as HIV reversetranscriptase, can block viral life-cycle. The sdAb of the invention canalso target intracellular viral proteins such as Ebola VP24 and thusblock Ebola's ability to shut down the host's anti-viral immuneresponse. The sdAbs of the invention can be used to target diseases whenthere is an overexpression of an intracellular molecule. Huntington'sdisease can be treated with sdAbs.

The sdAbs of the invention can be used with one or more compounds. Forexample, the sdAb of the invention can be used with JAK/STAT inhibitorssuch as, for example, Curcumin, Resveratrol, Cucurbitacin A, B, E, I, Q,Flavopiridol, Deoxytetrangomycin, Cyclopentenone derivatives,N-Acylhomoserine Lactone, Indirubin derivatives, Meisoindigo,Tyrphostins, Platinum-containing compounds (e.g., IS3-295),Peptidomimetics, antisense oligonucleotides, S3I-201, phosphotyrosintripeptide derivatives, HIV protease inhibitors (e.g., nelfinavir,indinavir, saquinavir, & ritornavir), JSI-124, XpYL, Ac-pYLPQTV-NH2, ISS610, CJ-1383, pyrimethamine, Metformin, Atiprimod, S3I-M2001, STX-0119;N-[2-(1,3,4-oxadiazolyl)]-4 quinolinecarboxamide derivative, S3I-1757,LYS;5,8-dioxo-6(pyridin-3-ylamino)-5,8,-dihydro-naphthalene-1-sulfonamide,withacinstin, Stattic, STA-21, LLL-3, LLL12, XZH-5, SF-1066, SF-1087,17o, Cryptotanshinone, FLL32, FLL62, C188-9, BP-1108 and BP-1075,Galiellalactone, JQ1, 5, 15 DPP, WP1066, Niclosamide, SD1008,Nifuroxazide, Cryptotanshinone, BBI quinone, and Ruxolitnib Phosphate.The one or more compounds can increase the therapeutic response andaugment the effectiveness of the sdAb of the invention. In addition, theeffectiveness of the sdAb can be increased by combining it withpeptides, peptidomimetics, and other drugs, such as, for example, butnot limited to, cimetidine, atorvastatin, celecoxib, metformin, andcimetidine. In addition, anti-STAT3 sdAbs can convert radioresistantcancers to radiosensitive cancers with respect to radiation therapy.

It is also contemplated that one or more sdAbs of the invention can becombined, or the sdAbs of the invention can be combined with othersdAbs.

It is contemplated that certain sdAbs of the invention can cross thecell membrane and enter the cell without the aid of additional targetingprotein sequences on the sdAb, and without the aid of exogenouscompounds that direct the sdAb to bind to the cell surface receptors andcross the cell membrane.

After crossing the cell membrane, these sdAbs can target transmembraneor intracellular molecules or antigens. These intracellular ortransmembrane targets can be, for example, proteins, carbohydrates,lipids, nucleic acids, mutated proteins, viral proteins, and prions. ThesdAb targets may function as enzymes, structural proteins of the cell,intracellular portions of cell membrane molecules, molecules within themembranes of organelles, any type of RNA molecule, any regions of DNA orchromosome, methylated or unmethylated nucleic acids, partiallyassembled molecules within the synthesis mechanism of the cell, secondmessenger molecules, and molecules within cell signaling mechanisms.Targets may include all molecules in the cytoplasm, nucleus, organelles,and cell membrane. Molecules destined for secretion or placement in thecell membrane can be targeted within the cytoplasm before leaving thecell.

The sdAb targets can be in humans, animals, plants, fungi, parasites,protists, bacteria, viruses, prions, prokaryotic cells, and eukaryoticcells. Some examples of inter- and intracellular signaling molecules andprotein groups that can be targeted by the sdAbs of the invention are:oncogene products, hormones, cytokines, growth factors,neurotransmitters, kinases (including tyrosine kinase, serine kinase,and threonine kinase), phosphatases, ubiquitin, cyclic nucleotides,cyclases (adenylyl and guanylyl), G proteins, phosphodiesterases, GTPasesuperfamily, immunoglobulins (antibodies, Fab fragments, binders,sdAbs), immunoglobulin superfamily, inositol phosphate lipids, steroidreceptors, calmodulin, CD group (e.g., CD4, CD8, CD28, etc.),transcription factors, TGF-beta, TNF-alpha and beta, TNF ligandsuperfamily, notch receptor signaling molecules, hedgehog receptorsignaling molecules, Wnt receptor signaling molecules, toll-likereceptor signaling molecules, caspases, actin, myosin, myostatin,12-lipoxygenase, 15-lipoxygenase, lipoxygenase superfamily, reversetranscriptase, viruses and their proteins, amyloid proteins, collagen, Gprotein coupled receptors, mutated normal proteins, prions, Ras, Raf,Myc, Src, BCR/ABL, MEK, Erk, Mos, Tp12, MLK3, TAK, DLK, MKK, p38, MAPK,MEKK, ASK, SAPK, JNK, BMK, MAP, JAK, PI3K, cyclooxygenase, STAT1, STAT2,STAT3, STAT4, STAT5a, STAT5b, STATE, Myc, p53, BRAF, NRAS, KRAS, HRASand chemokines.

KRAS is a Kirsten ras oncogene homolog from the mammalian ras genefamily. KRAS encodes a protein that is a member of the small GTPasesuperfamily. The protein is implicated in various malignancies,including lung adenocarcinoma, mucinous adenoma, ductal carcinoma of thepancreas, and colorectal carcinoma. Under normal conditions, Ras familymembers influence cell growth and differentiation events in asubcellular membrane compartmentalization-based signaling system.However, oncogenic Ras can deregulate processes that control both cellproliferation and apoptosis.

Anti-KRAS sdAbs were developed to target wild-type and mutated KRAS(G12D) in order to disrupt its role in malignant cells such as, forexample, cells involved in colorectal cancer, pancreatic cancer, biliarytract cancer, lung cancer, leukemias, and other metastatic malignancies.Without being bound by a particular mechanism, it is thought that theanti-KRAS sdAb binds KRAS and blocks the downstream signaling of KRAS inmalignant cells. Additionally, the anti-KRAS sdAb may successfully treatmalignancies that are resistant to anti-EGFR biologics (e.g., cetuximaband panitumumab).

Using methods that are well-known in the art, recombinant human mutantKRAS (G12D) protein was used to generate sdAbs that are directed againstor can bind to an epitope of KRAS or mutant KRAS (G12D), or other KRASmutants. Additionally, sdAbs can be generated to other KRAS mutants. Togenerate the anti-KRAS sdAbs, recombinant full-length human KRAS (GeneID: 3845) was expressed in Escherichia coli.

Several sdAbs were obtained and screened. The DNA sequence of oneanti-KRAS (G12D) sdAb, named KRAS_13 (SEQ ID NO:1), is shown below:

5′Gaggtgcagctggtggagtctgggggaggctcggtgcagactggagggtctctgagactctcctgtgcagtttctggaaatatcggcagcagctactgcatgggctggttccgccaggctccagggaagaagcgcgaggcggtcgcacgtattgtacgtgatggtgccactggctacgcagactacgtgaagggccgattcaccatctcccgagacagcgccaagaacactctgtatctgcaaatgaacaggctgatacctgaggacactgccatctactactgtgcggcagacctgcccccaggttgtttgactcaggcgatttggaattttggttatcggggccagggaaccctggtcaccgtctcctca-3′

The amino acid sequence of the anti-KRAS (G12D) sdAb (SEQ ID NO. 2),KRAS_13, is shown below, with the CDRs underlined:

EVQLVESGGGSVQTGGSLRLSCAVSGNIGSSYCMGWFRQAPGKKREAVARIVRDGATGYADYVKGRFTISRDSAKNTLYLQMNRLIPEDTAIYYCAADLPPGCLTQAIWNFGYRGQGTLVTVSS

Additionally, the present invention comprises one or more mousemonoclonal antibodies which are directed against one or more domains ofthe anti-KRAS sdAb of the invention. The mouse monoclonal antibody canbe generated by methods that are known by one of skill in the art, forexample, the mouse monoclonal antibody can be produced by a mousehybridoma. The mouse monoclonal antibody can be used in diagnosticassays, for example, the antibody can be used in an immunoassay such asan ELISA or mass spectrometry assay in order to measure the amount ofanti-KRAS sdAb present in a patient's serum. The cytotoxicity of KRAS(G12D) sdAbs on PANC-1 human pancreatic cancer cells was tested, asdescribed below.

STAT3 is a member of the signal transducers and activators oftranscription (STAT) family of proteins that carry both signaltransduction and activation of transcription functions. STAT3 is widelyexpressed and becomes activated through phosphorylation on tyrosineand/or serine as a DNA binding protein in response to a variouscytokines and growth factors such as EGF, IL-6, PDGF, IL-2 and G-CSF.The STAT3 phosphoprotein forms homodimers and heterodimers with othermembers of the STAT family and translocates to the nucleus in order tomodulate the transcription of various genes, and as a result plays a keyrole in many cellular processes such as cell growth, apoptosis,angiogenesis, immune evasion, and survival.

An anti-STAT3 sdAb can be given to patients and other organisms to treatdiseases caused by phosphorylated and non-phosphorylated STAT3, as wellas to prevent the development of disease or recurrence of disease. Forexample, patients who have undergone organ transplant and bone marrowtransplant are at higher risk for cutaneous SCCA and BCCA due to theimmunosuppressive medications they take. Administration of an anti-STAT3sdAb can reduce or eliminate this risk. Patients treated for amalignancy who are at risk for recurrence will benefit from treatmentwith the anti-STAT3 sdAb. Based on family medical history and HLA-type,some individuals will be at increased risk for some types of autoimmunediseases and may benefit from treatment with sdAbs to reduce risk ofdeveloping that autoimmune disease. Breast cancer risk can be reducedwith administration of anti-STAT3 medication such as GLG-302, asdemonstrated in a recent NCI study.

In addition to inhibiting STAT3, the anti-STAT3 sdAb can also inhibitSTAT1, STAT2, STAT4, STAT5a, STAT5b, and STATE due to the high degree ofhomology between these molecules.

Recombinant human STAT3 protein was used to produce anti-STAT sdAbs thatwere directed against or can bind to an epitope of STAT3. To generatethe anti-STAT3 sdAbs, recombinant full-length human STAT3 (Gene ID:6774) was expressed by baculovirus in Sf9 insect cells. The anti-STATsdAbs were cloned into vectors that can be expressed in both bacterialand mammalian cells, as shown in FIGS. 1 and 2.

The anti-STAT3 sdAb of the invention can be used to target STAT3 and allother STAT molecules inside the cell in order to inhibit cell growth,such as, for example, suppression of cancer cell growth. In addition,the anti-STAT3 sdAb can inhibit cell growth in other proliferativediseases such as psoriasis and macular degeneration via VEGF.

Without being limited to a particular mechanism of action, it is thoughtthat anti-STAT3 sdAb can eliminate cancer induced immune suppression bydecreasing STAT3 levels in antigen presenting cells such as, forexample, host dendritic cells. STAT3 inhibition promotes anti-cancerresponse by patient's innate and adaptive immune systems (i.e.,dendritic cells, macrophages, neutrophils, T cells, NK cells, and Bcells).

Using methods that are well known in the art, several anti-STAT sdAbswere obtained and screened for the ability to suppress cancer cellgrowth and induce apoptosis in cancer cell lines, as described below.The cytotoxicity and anti-proliferative activities of the anti-STAT3sdAbs was tested. In addition, the tolerance of anti-STAT3 sdAbs wastested in vitro and in vivo. The production of mouse monoclononalantibody directed against one or more domains of the anti-STAT sdAbs isdescribed below.

The amino acid sequence of one anti-STAT3 sdAb, named VHH13 (SEQ ID NO.3), is shown below:

HVQLVESGGGSVQAGGSLRLSCAASGANGGRSCMGWFRQVPGKEREGVSGISTGGLITYYADSVKGRFTISQDNTKNTLYLQMNSLKPEDTAMYYCATSRFDCYRGSWFNRYMYNSWGQGTQVTVSSThe three CDRs are underlined.

The amino acid sequence of a second anti-STAT3 sdAb, named VHH14 (SEQ IDNO. 4), is shown below:

QVQLVESGGGSVQAGGSLRLSCVASTYTGCMGWFRQAPGKEREGVAALSSRGFAGHYTDSVKGRFSISRDYVKNAVYLQMNTVKPEDAAMYYCAAREGWECGETWLDRTAGGHTYWGQGTLVTVSS

Again, the three CDRs are underlined. The protein sequences of otheranti-STAT3 sdAbs that were obtained are as follows:

STAT3_10 (SEQ ID NO. 5):(1) DVQLVESGGGSVQAGGSLRLSCVASTYTGCMGWFRQAPGKEREGVA A(48) LSSRGFAGHYTDSVKGRFSISRDYVKNAVYLQMNTVKPEDAAMYY CAARE(98) GWECGETWLDRTAGGHTYWGQGTQVTVSS STAT3_34 (SEQ ID NO. 6):(1) DVQLVESGGGSVQAGGSLRLSCVASTYTGCMGWFRQAPGKEREGVA A(48) LSSRGFAGHYTDSVKGRFSISRDYVKNAVYLQMNTVKPEDAAMYY CAARE(98) GWECGETWLDRTAGGHTYWGQGTQVTVSS STAT3_19 (SEQ ID NO. 7):(1) HVQLVESGGGSVQAGGSLRLSCVASTYTGCMGWFRQAPGKEREGVA A(48) LSSRGFAGHYTDSVKGRFSISRDYVKNAVYLQMNTVKPEDAAMYY CAARE(98) GWECGETWLDRTAGGHTYWGQGTQVTVSS STAT3_14 (SEQ ID NO. 8):(1) QVQLVESGGGSVQAGGSLRLSCVASTYTGCMGWFRQAPGKEREGVA A(48) LSSRGFAGHYTDSVKGRFSISRDYVKNAVYLQMNTVKPEDAAMYY CAARE(98) GWECGETWLDRTAGGHTYWGQGTLVTVSS STAT3_35 (SEQ ID NO. 9):(1) QVQLVESGGGSVQAGGSLRLSCVASTYTGCMGWFRQAPGKEREGVA A(48) LSSRGFAGHYTDSVKGRFSISRDYVKNAVYLQMNTVKPEDAAMYY CAARE(98) GWECGETWLDRTAGGHTYWGQGTLVTVSS STAT3_9 (SEQ ID NO. 10):(1) QVQLVESGGGSVQAGGSLRLSCVASTYTGCMGWFRQAPGKEREGVA A(48) LSSRGFAGHYTDSVKGRFSISRDYVKNAVYLQMNTVKPEDAAMYY CAARE(98) GWECGETWLDRTAGGHTYWGQGTLVTVSS STAT3_30 (SEQ ID NO. 11):(1) QVQLVESGGGSVQAGGSLRLSCVASTYTGCMGWFRQAPGKEREGVA A(48) LSSRGFAGHYTDSVKGRFSISRDYVKNAVYLQMNTVKPEDAAMYY CAARE(98) GWECGETWLDRTAGGHTYWGQGTLVTVSS STAT3_23 (SEQ ID NO. 12):(1) QVQLVESGGGSVQAGGSLRLSCVASTYTGCMGWFRQAPGKEREGVA A(48) LSSRGFAGHYTDSVKGRFSISRDYVKNAVYLQMNTVKPEDAAMYY CAARE(98) GWECGETWLDRTAGSHTYWGQGTLVTVSS STAT3_24 (SEQ ID NO. 13):(1) EVQLVESGGGSVQAGGSLRLSCVASTYTGCMGWFRQAPGKEREGVA A(48) LSSRGFAGHYTDSVKGRFSISRDYVKNAVYLQMNTVKPEDAAMYY CAARE(98) GWECGETWLDRTAGGHTYWGQGTLVTVSS STAT3_36 (SEQ ID NO. 14):(1) DVQLVESGGGSVQAGDSLRLSCVASTYTGCMGWFRQAPGKEREGVA A(48) LSSRGFAGHYTDSVKGRFSISRDYVKNAVYLQMNTVKPEDAAMYY CAARE(98) GWECGETWLDRTAGGHTYWGQGTLVTVSS STAT3_12 (SEQ ID NO. 15):(1) QVQLVESGGGSVQAGGSLRLSCAASGANGGRSCMGWFRQVPGKERE GVSG(51) ISTGGLITYYADSVKGRFTISQDNTKNTLYLQMNSLKPEDTAMYY CATSR(101) FDCYRGSWFNRYMYNSWGQGTLVTVSS STAT3_16 (SEQ ID NO. 16):(1) QVQLVESGGGSVQAGGSLRLSCAASGANGGRSCMGWFRQVPGKERE GVSG(51) ISTGGLITYYADSVKGRFTISQDNTNNTLYLQMNSLKPEDTAMYY CATSR(101) FDCYRGSWFNRYMYNSWGQGTLVTVSS STAT3_11 (SEQ ID NO. 17):(1) EVQLVESGGGSVQAGGSLRLSCAASGANGGRSCMGWFRQVPGKERE GVSG(51) ISTGGLITYYADSVKGRFTISQDNTKNTLYLQMNSLKPEDTAMYY CATSR(101)FDCYRGSWFNRYMYNSWGQGTLVTVSS STAT3_20 (SEQ ID NO. 18):(1) DVQLVESGGGSVQAGGSLRLSCAASGANGGRSCMGWFRQVPGKERE GVSG(51) ISTGGLITYYADSVKGRFTISQDNTKNTLYLQMNSLKPEDTAMYY CATSR(101) FDCYRGSWFNRYMYNSWGQGTLVTVSS STAT3_2 (SEQ ID NO. 19):(1) DVQLVESGGGSVQAGGSLRLSCAASGANGGRSCMGWFRQVPGKERE GVSG(51) ISTGGLITYYADSVKGRFTISQDNTKNTLYLQMNSLKPEDTAMYY CATSR(101) FDCYRGSWFNRYMYNSWGQGTLVTVSS STAT3_15 (SEQ ID NO. 20):(1) DVQLVESGGGSVQAGGSLRLSCAASGANGGRSCMGWFRQVPGKERE GVSG(51) ISTGGLITYYADSVKGRFTISQDNTKNTLYLQMNSLKPEDTAMYY CATSR(101) FDCYRGSWFNRYMYNSWGQGTLVTVSS STAT3_6 (SEQ ID NO. 21):(1) HVQLVESEGGSVQAGGSLRLSCAASGANGGRSCMGWFRQVPGKERE GVSG(51) ISTGGLITYYADSVKGRFTISQDNTKNTLYLQMNSLKPEDTAMYY CATSR(101) FDCYRGSWFNRYMYNSWGQGTLVTVSS STAT3_33 (SEQ ID NO. 22):(1) QVQLVESGGGSVQAGGSLRLSCAASGANGGRSCMGWFRQVPGKERE GVSG(51) ISTGGLITYYADSVKGRFTISQDNTKNTLYLQMNSLKPEDTAMYY CATSR(101) FDCYRGSWFNRYMYNSWGQGTQVTVSS STAT3_17 (SEQ ID NO. 23):(1) QVQLVESGGGSVQAGGSLRLSCAASGANGGRSCMGWFRQVPGKERE GVSG(51) ISTGGLITYYADSVKGRFTISQDNTKNTLYLQMNSLKPEDTAMYY CATSR(101) FDCYRGSWFNRYMYNSWGQGTQVTVSS STAT3_25 (SEQ ID NO. 24):(1) EVQLVESGGGS VQAGGSLRLSCAASGANGGRSCMGWFRQVPGKER EGVSG(51) ISTGGLITYYADSVKGRFTISQDNTKNTLYLQMSSLKPEDTAMYY CATSR(101) FDCYRGSWFNRYMYNSWGQGTQVTVSS STAT3_32 (SEQ ID NO. 25):(1) DVQLVESGGGSVQAGGSLRLSCAASGANGGRSCMGWFRQVPGKERE GVSG(51) ISTGGLITYYADSVKGRFTISQDNTKNTLYLQMNSLKPEDTAMYY CATSR(101) FDCYRGSWFNRYMYNSWGQGTQVTVSS STAT3_13 (SEQ ID NO. 26):(1) HVQLVESGGGSVQAGGSLRLSCAASGANGGRSCMGWFRQVPGKERE GVSG(51) ISTGGLITYYADSVKGRFTISQDNTKNTLYLQMNSLKPEDTAMYY CATSR(101) FDCYRGSWFNRYMYNSWGQGTQVTVSS STAT3_39 (SEQ ID NO. 27):(1) HVQLVESGGGSVQAGGSLRLSCAASGANGGRSCMGWFRQVPGKERE GVSG(51) ISTGGLITYYADSVKGRFTISQDNTKNTLYLQMNSLKPEDTAMYY CATSR(101) FDCYRGSWFNRYMYNSWGQGTQVTVSS STAT3_4 (SEQ ID NO. 28):(1) HVQLVESGGGSVQAGGSLRLSCAASGANGGRSCMGWFRQVPGKERE GVSG(51) ISTGGLITYYADSVKGRFTISQDNTKNTLYLQMNSLKPEDTAMYY CATSR(101) FDCYRGSWFNRYMYNSWGQGTQVTVSS STAT3_29 (SEQ ID NO. 29):(1) HVQLVESGGGSVQAGGSLRLSCAASGANGGRSCMGWFRQVPGKERE GVSG(51) ISTGGLITYYADSVKGRFTISQDNTKNTLYLQMNSLKPEDTAMYY CATSR(101) FDCYRGSWFNRYMYNSWGQGTQVTVSS

The corresponding anti-STAT3 DNA sequences are as follows:

Stat3_VHH-10 (SEQ ID NO. 30):5′-gatgtgcagctggtggagtctgggggaggctcggtgcaggctggaggctctctgagactctcctgtgtagcctctacatacaccggctgcatgggctggttccgccaggctcctggaaaggagcgcgagggagtcgcagctcttagtagccgtggttttgccgggcactataccgactccgtgaagggccgattctccatctcccgagactacgtcaagaatgcggtgtatctgcaaatgaacactgtgaaacctgaggacgctgccatgtactactgtgcagcacgggagggatgggagtgcggtgagacctggttggaccggaccgccgggggccatacctactggggccaggggacccaggtcaccgtctcctca-3′ Stat3_VHH-14 (SEQ ID NO. 31):5′-caggtgcagctggtggagtctgggggaggctcggtgcaggctggaggctctctgagactctcctgtgtagcctctacatacaccggctgcatgggctggttccgccaggctcctggaaaggagcgcgagggagtcgcagctcttagtagccgtggttttgccgggcactataccgactccgtgaagggccgattctccatctcccgagactacgtcaagaatgcggtgtatctgcaaatgaacactgtgaaacctgaggacgctgccatgtactactgtgcagcacgggagggatgggagtgcggtgagacctggttggaccggaccgccgggggccatacctactggggccaggggaccctggtcaccgtctcctca-3′ Stat3_VHH-12 (SEQ ID NO. 32):5′-caggtgcagctggtggagtctgggggaggctcggtgcaggctggagggtctctgagactctcctgtgcagcctctggagccaatggtggtcggagctgcatgggctggttccgccaggttccagggaaggagcgcgagggggtttctggtatttcaaccggtggtcttattacatactatgccgactccgtgaagggccgattcaccatctcccaagacaacaccaagaacacgctgtatctgcaaatgaacagcctgaaacctgaggacactgccatgtactactgtgcgacgagtcggtttgactgctatagaggctcttggttcaaccgatatatgtataacagttggggccaggggaccctggtcaccgtctcctca-3′ Stat3_VHH-13 (SEQ ID NO. 33):5′-catgtgcagctggtggagtctgggggaggctcggtgcaggctggagggtctctgagactctcctgtgcagcctctggagccaacggtggtcggagctgcatgggctggttccgccaggttccagggaaggagcgcgagggggtttctggtatttcaaccggtggtcttattacatactatgccgactccgtgaagggccgattcaccatctcccaagacaacaccaagaacacgctgtatctgcaaatgaacagcctgaaacctgaggacactgccatgtactactgtgcgacgagtcggtttgactgctatagaggctcttggttcaaccgatatatgtataacagttggggccaggggacccaggtcactgtctcctca-3′ Stat3_VHH-20 (SEQ ID NO. 34):5′-gatgtgcagctggtggagtctgggggaggctcggtgcaggctggagggtctctgagactctcctgtgcagcctctggagccaatggtggtcggagctgcatgggctggttccgccaggttccagggaaggagcgcgagggggtttctggtatttcaaccggtggtcttattacatactatgccgactccgtgaagggccgattcaccatctcccaagacaacaccaagaacacgctgtatctgcaaatgaacagcctgaaacctgaggacactgccatgtactactgtgcgacgagtcggtttgactgctatagaggctcttggttcaaccgatatatgtataacagttggggccaggggaccctggtcaccgtctcctca-3′ Stat3_VHH-23 (SEQ ID NO. 35):5′-caggtgcagctggtggagtctgggggaggctcggtgcaggctggaggctctctgagactctcctgtgtagcctctacatacaccggctgcatgggctggttccgccaggctcctggaaaggagcgcgagggagtcgcagctcttagcagccgtggttttgccgggcactataccgactccgtgaagggccgattctccatctcccgagactacgtcaagaatgcggtgtatctgcaaatgaacactgtgaaacctgaggacgctgccatgtactactgtgcagcacgggagggatgggagtgcggtgagacctggttggaccggaccgccgggagccatacctactggggccaggggaccctggtcaccgtctcctca-3′ Stat3_VHH-24 (SEQ ID NO. 36):5′-gaggtgcagctggtggagtctgggggaggctcggtgcaggctggaggctctctgagactctcctgtgtagcctctacatacaccggctgcatgggctggttccgccaggctcctggaaaggagcgcgagggagtcgcagctcttagtagccgtggttttgccgggcactataccgactccgtgaagggccgattctccatctcccgagactacgtcaagaatgcggtgtatctgcaaatgaacactgtgaaacctgaggacgctgccatgtactactgtgcagcacgggagggatgggagtgcggtgagacctggttggaccgaaccgccgggggccatacctactggggccaggggaccctggtcaccgtctcctca-3′ Stat3_VHH-25 (SEQ ID NO. 37):5′-gaggtgcagctggtggagtctgggggaggctcggtgcaggctggagggtctctgagactctcctgtgcagcctctggagccaatggtggtcggagctgcatgggctggttccgccaggttccagggaaggagcgcgagggggtttctggtatttcaaccggtggtcttattacatactatgccgactccgtgaagggtcgattcaccatctcccaagacaacaccaagaacacgctgtatctgcaaatgagcagcctgaaacctgaggacactgccatgtactactgtgcgacgagtcggtttgactgctatagaggctcttggttcaaccgatatatgtataacagttggggccaggggacccaggtcaccgtctcctca-3′ Stat3_VHH-19 (SEQ ID NO. 38):5′-catgtgcagctggtggagtctggggggggctcggtgcaggctggaggctctctgagactctcctgtgtagcctctacatacaccggctgcatgggctggttccgccaggctcctggaaaggagcgcgagggagtcgcagctcttagtagccgtggttttgccgggcactataccgactccgtgaagggccgattctccatctcccgagactacgtcaagaatgcggtgtatctgcaaatgaacactgtgaaacctgaggacgctgccatgtactactgtgcagcacgggagggatgggagtgcggtgagacctggttggaccggaccgccgggggccatacctactggggccaggggacccaggtcaccgtctcctca-3′ Stat3_VHH-32 (SEQ ID NO. 39):5′-gatgtgcagctggtggagtctgggggaggctcggtgcaggctggagggtctctgagactctcctgtgcagcctctggagccaatggtggtcggagctgcatgggctggttccgccaggttccagggaaggagcgcgagggggtttctggtatttcaaccggtggtcttattacatactatgccgactccgtgaagggccgattcaccatctcccaagacaacaccaagaacacgctgtatctgcaaatgaacagcctgaaacctgaggacactgccatgtactactgtgcgacgagtcggtttgactgctatagaggctcttggttcaaccgatatatgtataacagttggggccaggggacccaggtcaccgtctcctca-3′ Stat3_VHH-33 (SEQ ID NO. 40):5′-caggtgcagctggtggagtctgggggaggctcggtgcaggctggagggtctctgagactctcctgtgcagcctctggagccaatggtggtcggagctgcatgggctggttccgccaggttccagggaaggagcgcgagggggtttctggtatttcaaccggtggtcttattacatactatgccgactccgtgaagggccgattcaccatctcccaagacaacaccaagaacacgctgtatctgcaaatgaacagcctgaaacctgaggacactgccatgtactactgtgcgacgagtcggtttgactgctatagaggctcttggttcaaccgatatatgtataacagttggggccaggggacccaggtcaccgtctcctca-3′ Stat3_VHH-36 (SEQ ID NO. 41):5′-gatgtgcagctggtggagtctgggggaggctcggtgcaggctggagactctctgagactctcctgtgtagcctctacatacaccggctgcatgggctggttccgccaggctcctggaaaggagcgcgagggagtcgcagctcttagtagccgtggttttgccgggcactataccgactccgtgaagggccgattctccatctcccgagactacgtcaagaatgcggtgtatctgcaaatgaacactgtgaaacctgaggacgctgccatgtactactgtgcagcacgggagggatgggagtgcggtgagacctggttggaccggaccgccgggggccatacctactggggccaggggaccctggtcactgtctcctca-3′ Stat3_VHH-11 (SEQ ID NO. 42):5′-gtgcagctggtggagtctgggggaggctcggtgcaggctggagggtctctgagactctcctgtgcagcctctggagccaatggtggtcggagctgcatgggctggttccgccaggttccagggaaggagcgtgagggggtttctggtatttcaaccggtggtcttattacatactatgccgactccgtgaagggccgattcaccatctcccaagacaacaccaagaacacgctgtatctgcaaatgaacagcctgaaacctgaggacactgccatgtactactgtgcgacgagtcggtttgactgctatagaggctcttggttcaaccgatatatgtataacagttggggccaggggaccctggtcactgtctcctca-3′ Stat3_VHH-6 (SEQ ID NO. 43):5′-gtgcagctggtggagtctgagggaggctcggtgcaggctggagggtctctgagactctcctgtgcagcctctggagccaatggtggtcggagctgcatgggctggttccgccaggttccagggaaggagcgcgagggggtttctggtatttcaaccggtggtcttattacatactatgccgactccgtgaagggccgattcaccatctcccaagacaacaccaagaacacgctgtatctgcaaatgaacagcctgaaacctgaggacactgccatgtactactgtgcgacgagtcggtttgactgctatagaggctcttggttcaaccgatatatgtataacagttggggccaggggaccctggtcaccgtctcctca-3′ Stat3_VHH-1 (SEQ ID NO. 44):5′-gtgcagctggtggagtctgggggaggctcggtgcaggctggagggtctctgagactctcctgtgcagcctctggagccaatggtggtcggagctgcatgggctggttccgccaggttccagggaaggagcgcgagggggtttctggtatttcaaccggtggtcttattacatactatgccgactccgtgaagggccgattcaccatctcccaagacaacaccaataacacgctgtatctgcaaatgaacagcctgaaacctgaggacactgccatgtactactgtgcgacgagtcggtttgactgctatagaggctcttggttcaaccgatatatgtataacagttggggccaggggaccctggtcactgtctcctca-3′

Additionally, the present invention comprises one or more mousemonoclonal antibodies which are directed against one or more domains ofthe anti-STAT3 sdAb of the invention. The mouse monoclonal antibody canbe generated by methods that are known by one of skill in the art, forexample, the mouse monoclonal antibody can be produced by a mousehybridoma. The mouse monoclonal antibody can be used in diagnosticassays, for example, the antibody can be used in an immunoassay such asan ELISA in order to measure the amount of anti-STAT3 sdAb present in apatient's serum. It should be appreciated that the method is not limitedto anti-STAT3 sdAbs, and could be used to produce a mouse antibodydirected towards any of the sdAbs of the present invention.

The TNF-alpha gene encodes a multifunctional proinflammatory cytokinethat belongs to the tumor necrosis factor (TNF) superfamily. Thiscytokine is mainly secreted by macrophages. The cytokine is involved inthe regulation of a wide spectrum of biological processes includinggrowth regulation, differentiation, inflammation, viral replication,tumorigenesis, and autoimmune diseases; and in viral, bacterial, fungal,and parasitic infections. Besides inducing hemorrhagic necrosis oftumors, TNF was found to be involved in tumorigenesis, tumor metastasis,viral replication, septic shock, fever, inflammation, cachexia, andautoimmune diseases including Crohn's disease, and rheumatoid arthritisas well as graft-versus-host disease.

The present invention provides sdAbs, proteins, and polypeptides thatare directed against TNF-alpha, in particular against human TNF-alphainside the cell or cell membrane, so as to prevent the secretion ofTNF-alpha by cells.

It is contemplated that the anti-TNF-alpha sdAbs and polypeptides of theinvention can be used for the prevention and/or treatment of diseasesand disorders associated with and/or mediated by TNF-alpha, such asinflammation, rheumatoid arthritis, Crohn's disease, ulcerative colitis,inflammatory bowel syndrome, multiple sclerosis, Addison's disease,autoimmune hepatitis, autoimmune parotitis, diabetes type 1,epididymitis, glomerulonephritis, Graves' disease, Guillain-Barresyndrome, Hashimoto's disease, hemolytic anemia, systemic lupuserythematosus, male infertility, multiple sclerosis, myasthenia gravis,pemphigus, psoriasis, rheumatic fever, rheumatoid arthritis,sarcoidosis, scleroderma, Sjogren's syndrome, spondyloarthropathies,thyroiditis, vasculitis, and weight loss due to cancer and cachexia.

TNF-alpha exists in different forms; there are monomeric and multimericforms, including a trimeric form. It is within the scope of theinvention that the sdAbs, proteins and polypeptides of the inventionbind to TNF-alpha in its different form, i.e., monomeric form ormultimeric forms. Thus, when sdAbs, proteins and polypeptides of theinvention are directed to TNF-alpha, it should be understood that thisalso comprises sdAbs, proteins and polypeptides directed againstTNF-alpha in its trimeric form.

It is known that signal transduction by TNF involves crosslinking by TNFreceptors by a trimer of TNF molecules, which contains three receptorbinding sites (see, for example, Peppel et al., J. Exp. Med., 174(1991), 1483-1489).

Recombinant human TNF-alpha protein was used to generate sdAbs that aredirected against or can bind to an epitope of TNF-alpha. To generate theanti-TNF-alpha sdAbs, recombinant full-length human TNF-alpha (Gene ID:7124) was expressed in Escherichia coli and used as the target antigen.

Thirty-five sdAbs against the TNF-alpha protein were obtained. Theseanti-TNF-alpha antibodies were divided into three groups based onsequence homology.

The amino acid sequence of the first anti-TNF-alpha sdAb, namedTNF-alpha VHH66 (SEQ ID NO. 45) sdAb, is shown below:

HVQLVESGGGSVEAGGSLRLSCAASGFRYAAYCMGWFRQADGKEREGVATIDIDGLTTHADSVKGRFTISRDNAKNTLSLQMNDLKPEDTAMYYCAADRDRCGSIWTYAYKYRG-QGTLVTVSSThe three CDRs are underlined.

In The amino acid sequence of the second anti-TNF-alpha sdAb, namedTNF-alpha VHH69 (SEQ ID NO. 46) sdAb, is shown below:

EVQLVESGGGSVLAGGSLRLSCVASGFTSRYNYMAWFRQAPGKEREGVATIGTASGSADYYGSVKDRFTISQDNAKNTVYLQMNSLKPEDTAMYYCAARTYGTISLTPSDYRYWGQGTLVTVSSThe three CDRs are underlined.

The amino acid sequence of the third anti-TNF-alpha sdAb, namedTNF-aphha VHH62 (SEQ ID NO. 47) sdAb, is shown below:

QVQLVESGGGPVQAGETLRLSCTASGFTFAEADMGWYRQAPGHECELVSTITTEGITSEASSYYADSVRGRFTISRDNAKNMVYLQMNSLKPEDTAVYYCAPDPYAYSTYSDYCSWAQGTQGTLVTVSS

The three CDRs are underlined. Other anti-TNF-alpha sdAbs that werefound include the sequences below, again with the CDRs underlined:

TNF_2 (SEQ ID NO. 48): QVQLVESGGGSVEAGRSLRLSCAASGFRYAAYCMGWFRQADGKEREGVATIDIDGQTTHADSVKGRFTISRDNAKNTLSLQMNDLKPEDTAMYYCAADRDRCGSIWTYAYKYRGQGTQVTVSS TNF_46 (SEQ ID NO. 49):QVQLVESGGGSVEAGGSLRLSCAASGFRYAAYCMGWFRQADGKEREGVATIDIDGQTTHADSVKGRFTISRDNVKNTLSLQMNDLKPEDTAMYYCAADRDRCGSIWTYAYKYRGQGTQVTVSS TNF_71 (SEQ ID NO. 50):QVQLVESGGGSVEAGGSLRLSCAASGFRYAAYCMGWFRQADGKEREGVATIDIDGLTTHADSVKGRFTISRDNAKNTLSLQMNDLKPEDTAMYYCAADRDRCGSIWTYAYKYRGQGTQVTVSS TNF_21 (SEQ ID NO. 51):QVQLVESGGGSVEAGGSLRLSCAASGFRYAAYCMGWFRQADGKEREGVATIDIDGQTTHADSVKGRFTISRDNAKNTLSLQMNDLKPEDTAMYYCAADRDRCGSIWTYAYKYRGQGTQVTVSS TNF_38 (SEQ ID NO. 52):EVQLVESGGGSVEAGGSLRLSCAASGFRYAAYCMGWFRQADGKEREGVATIDIDGQTTHADSVKGRFTISRDNAKNTLSLQMNDLKPEDTAMYYCAADRDRCGSIWTYAYKYRGQGTQVTVSS TNF_18 (SEQ ID NO. 53):EVQLVESGGGSVEAGGSLRLSCAASGFRYAAYCMGWFRQADGKEREGVATIDIDGLTTHADSVKGRFTISRDNAKNTLSLQMNDLKPEDTAMYYCAADRDRCGSIWTYAYKYRGQGTLVTVSS TNF_37 (SEQ ID NO. 54):DVQLVESGGGSVEAGGSLRLSCAASGFRYAAYCMGWFRQADGKEREGVATIDIDGQTTHADSVKGRFTISRDNAKNTLSLQMNDLKPEDTAMYYCAADRDRCGSIWTYAYKYRGQGTLVTVSS TNF_66 (SEQ ID NO. 55):HVQLVESGGGSVEAGGSLRLSCAASGFRYAAYCMGWFRQADGKEREGVATIDIDGLTTHADSVKGRFTISRDNAKNTLSLQMNDLKPEDTAMYYCAADRDRCGSIWTYAYKYRGQGTLVTVSS TNF_68 (SEQ ID NO. 56):HVQLVESGGGSVEAGGSLRLSCAASGFRYAAYCMGWFRQADGKEREGVATIDIDGLATHADSVKGRFTISRDNAKNTLSLQMNDLKPEDTAMYYCAADRDRCGSIWTYAYKYRGQGTLVTVSS TNF_78 (SEQ ID NO. 57):HVQLVESGGGSVEAGGSLRLSCAASGFRYAAYCMGWFRQADRKEREGVATIDIDGQTTHADSVKGRFTISRDNAKNTLSLQMNDLKPEDTAMYYCAADRDRCGSIWTYAYKYRGQGTQVTVSS TNF_67 (SEQ ID NO. 58):HVQLVESGGGSVQAGGSLRLSCAASGFRYAAYCMGWFRQADGKVREGVATIDIDGQTTHADSVKGRFTISRDNAKNTLSLQMNDLKPEDTAMYYCAADRDRCGSIWTYAYKYRGQGTLVTVSS TNF_6 (SEQ ID NO. 59):QVQLVESGGGSVQAGGSLRLSCAASGFIDSFGVMAWFRQAPGKEREGVAAVYRRAGDTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDSAMYYCAARTYGSVSSWTGYKYWGQGTQVTVSS TNF_7 (SEQ ID NO. 60):DVQLVESGGGSVQAGGSLRLSCAASGFIDSFGVMAWFRQTPGKEREGVAAVYRRAGDTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDSAMYYCAARTYGSVSSWTGYKYWGQGTQVTVSS TNF_13 (SEQ ID NO. 61):DVQLVESGGGSVQVGGSLTLSCAVSGYTDSYGVMAWFRQAPGKEREGVASIYRNSGITYYPDSVKGRFTISRDNAKNTVLLQMNSLKPEDSATYYCAVRSFGSVSTWAGYVYWGQGTQVTVSS TNF_60 (SEQ ID NO. 62):DVQLVESGGGSVQAGGSLRLSCAASGFIDSFGVMAWFRQAPGKEREGVAAVYRRAGDTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDSAMYYCAARTYGSVSSWTGYKYWGRGTQVTVSS TNF_73 (SEQ ID NO. 63):DVQLVESGGGSVRAGGSLRLSCTASGDTSKSDCMAWFRQAPGKERERVGAIYTRNGYTHYADSVNGRFTISQDNAKNALYLQMSGLKPEDTAMYYCAARFRIYGQCVEDDDIDYWGQGTLVTVSS TNF_69 (SEQ ID NO. 64):EVQLVESGGGSVLAGGSLRLSCVASGFTSRYNYMAWFRQAPGKEREGVATIGTASGSADYYGSVKDRFTISQDNAKNTVYLQMNSLKPEDTAMYYCAARTYGTISLTPSDYRYWGQGTLVTVSS TNF_76 (SEQ ID NO. 65):QVQVVEYGGGSVQAGETVRLSCTASGFTFAEADMGWYRQAPGHEWELVSNITTEGITSEASSSYADSVRGRFTIFDNAKNMVYLQMNSLKHEDTAVYYCAPDPYAYSTYREYCTWAQGTQGTLVTVSS TNF_62 (SEQ ID NO. 66):QVQLVESGGGPVQAGETLRLSCTASGFTFAEADMGWYRQAPGHECELVSTITTEGITSEASSYYADSVRGRFTISRDNAKNMVYLQMNSLKPEDTAVYYCAPDPYAYSTYSDYCSWAQGTQGTLVTVSS TNF_43 (SEQ ID NO. 67):QVQLVESGGGSVQAGETLRLSCTASGFTFAEADMGWYRQAPGHECELVSTITTEGITSEASSYYADSVRGRFTISRDNAKNMVYLQMNSLKPEDTAVYYCAPDPYAYSTYSDYCTWAQGTQGTLVTVSS TNF_15 (SEQ ID NO. 68):QVQPVESGGGSVQAGETLRLSCTASGFTFAEADMGWYRQAPGHECELVSTITTEGITSEASSYYADSVRGRFTISRDNAKNMVYLQMNSLKPEDTAVYYCAPDPYAYSTYSDYCTWAQGAQGTLVTVSS TNF_11 (SEQ ID NO. 69):QVQLVESGGGSVQAGETLRLSCTASGFTFAEADMGWYRQAPGHECELVSTITTEGITSEASSYYADSVRGRFTISRDNAKNMVYLQMNSLKPEDTAVYYCAPDPYAYSTYSDYCSWAQGTQGTQVTVSS TNF_17 (SEQ ID NO. 70):QVQLVESGGGSVQAGETLRLSCTASGFTFAEADMGWYRQAPGHECELVSTITTEGITSEASSYYADSVRGRFTISRDNAKNMVYLQMNSLKPEDTAVYYCAPDPYAYSTYSDYCTWAQGTQGTQVTVSS TNF_63 (SEQ ID NO. 71):QVQLVESGGGSVQAGETLRLSCTASGFTFAEADMGWYRQAPGHECELVSTITTEGITSEASSYYADSVRGRFTISRDNAKNMVYLQMNSLKPEDTAVYYCAPDPYAYSTYSDYCTWAQGTQGTLVTVSS TNF_20 (SEQ ID NO. 72):HVQLVESGGGSVQAGETLRLSCTASGFTFAEADMGWYRQAPGHECELVSTITTEGITSEASSYYADSVRGRFTISRDNAKNMVYLQMNSLKPEDTAVYYCAPDPYAYSTYSDYCTWAQGTQGTQVTVSS TNF_58 (SEQ ID NO. 73):EVQLVESGGGSVQAGETLRLSCTASGFTFAEADMGWYRQAPGHECELVSTITTEGITSEASSYYADSVRGRFTISRDNAKNMVYLQMNSLKPEDTAVYYCAPDPYAYSTYSDYCTWAQGTQGALVTVSS TNF_27 (SEQ ID NO. 74):EVQLVESGGGSVQAGETLRLSCTASGFTFAEADMGWYRQAPGHECELVSTITTEGITSEASSYYADSVRGRFTISRDNAKNMVYLQMNSLKPEDTAVYYCAPDPYAYSTYSDYCTWAQGTQGTLVTVSS TNF_28 (SEQ ID NO. 75):EVQLVESGGGSVQAGETLRLSCTASGFTFAEADMGWYRQAPGHECELVSTITTEGITSEASSYYADSVRGRFTISRDNAKNMVYLQMNSLKPEDTAVYYCAPDPYAYSTYSDYCSWAQGTQGTQVTVSS TNF_4 (SEQ ID NO. 76):EVQLVESGGGSVQAGETLRLSCTASGFTFAEADMGWYRQAPGHECELVSTITTEGITSEASSYYADSVRGRFTISRDNAKNMVYLQMNSLKPEDTAVYYCAPDPYAYSTYSDYCTWAQGTQGTQVTVSS TNF_14 (SEQ ID NO. 77):DVQLVESRGGSVQAGETLRLSCTASGFTFAEADMGWYRQAPGHECELVSTITTEGITSEASSYYADSVRGRFTISRDNAKNMVYLQMNSLKPEDTAVYYCAPDPYAYSTYSDYCTWAQGTQGTLVTVSS TNF_3 (SEQ ID NO. 78):DVQLVESGGGSVQAGETLRLSCTASGFTFAEADMGWYRQAPGHVCELVSTITTEGITSEASSYYADSVRGRFTISRDNAKNMVYLQMNSLKPEDTAVYYCAPDPYAYSTYSDYCSWAQGTQGTQVTVSS TNF_1 (SEQ ID NO. 79):DVQLVESGGGSVQAGETLRLSCTASGFTFAEADMGWYRQAPGLECELVSTITTEGITSEASSYADSVRGRFTISRDNAKNMVYLQMNSLKPEDTAVYYCAPDPYAYSTYSEYCTWAQGTQGTLVTVSS TNF_45 (SEQ ID NO. 80):DVQLVESGGGSVQAGETLRLSCTASGFTFAEADMGWYRQAPGHECELVSTITTEGITSEASSYYADSVRGRFTISRDNAKNMVYLQMNSLKPEDTAVYYCAPDPYAYSTYSDYCTWAQGTQGTLVTVSS TNF_22 (SEQ ID NO. 81):DVQLVESGGGSVQAGETLRLSCTASGFTFAEADMGWYRQAPGHECELVSTITTEGITSVASSYYADSVRGRFTISRDNAKNMVYLQMNSLKPEDTAVYYCAPDPYAYSTYSDYCTWAQGTQGTQVTVSS

The in vitro growth inhibition of several TNF-alpha sdAbs was tested, asdescribed below. Additionally, the present invention comprises one ormore mouse monoclonal antibodies which are directed against one or moredomains of the anti-TNF-alpha sdAb of the invention. The mousemonoclonal antibody can be generated by methods that are known by one ofskill in the art, as described above. The mouse monoclonal antibody canbe used in diagnostic assays, such as, for example, an immunoassay suchas an ELISA in order to measure the amount of anti-TNF-alpha sdAbpresent in a patient's serum.

The RAF proteins are a family of serine/threonine-specific kinases thatserve as a central intermediate in transmitting extracellular signals tothe mitogen-activated protein kinase cascade, which controls cellgrowth, differentiation and survival. BRAF is a member of the RAF familythat is activated by members of the Ras family upon growthfactor-induced stimulation. Active Ras can induce heterodimerization ofcRaf and BRAF and this may explain the observed cooperativity of cRafand BRaf in cells responding to growth factor signals. Activatingmutations in the BRAF gene are present in a large percentage of humanmalignant melanomas and in a proportion of colon cancers. The vastmajority of these mutations result in a valine to glutamic acid changeat residue 599 within the activation segment of BRAF.

Anti-BRAF sdAbs were developed to target wild-type and mutated BRAF inorder to disrupt its role in malignant cells such as, for example, cellsinvolved in colon cancer and other malignancies.

Using methods that are well-known in the art, recombinant human BRAFprotein was used to generate sdAbs that are directed against or can bindto an epitope of BRAF.

Additionally, the present invention comprises one or more mousemonoclonal antibodies which are directed against one or more domains ofthe anti-BRAF sdAb of the invention. The mouse monoclonal antibody canbe generated by methods that are known by one of skill in the art. Themouse monoclonal antibody can be used in diagnostic assays, for example,the antibody can be used in an immunoassay such as an ELISA in order tomeasure the amount of anti-BRAF sdAb present in a patient's serum.

EXAMPLES Example 1 Anti-STAT3 VHH13 (SEQ ID No. 3) sdAb Binds STAT3

In this example, the affinity of two VHH targets against STAT3 wasmeasured using Octet based label-free binding assay. Anti-STAT3 VHH13(SEQ ID NO:3) sdAb, anti-KRAS (negative control) and GST-STAT3 (16 kDamonovalent antigen, Creative BioMart #STAT3-1476H) were used as antigenprobes in this assay. The GST-STAT3 protein was captured at 20 μg/ml inPBS using aminopropylsilane (APS) dip and read biosensors, specificallymeant for hydrophobic protein. The probes were then dipped into wellswith the GST-STAT3 protein, anti-STAT3 VHH13 (SEQ ID NO:3) sdAb oranti-KRAS at a concentration as indicated. The association rate (onrate) of the antigen was measured. The sensors were quenched with 1% BSAin water. The probes were dipped into assay buffer (PBS) and thedissociation rate (off rate) was measured.

The affinity, represented by the equilibrium constant for thedissociation of an antigen with an antigen-binding protein (KD) wasdetermined from the obtained affinity constant (KA), and KD using 1:1global fit analysis Fortebio software as shown below in Table 1.Affinity was determined by averaging KD values for curves with R2values >0.95. The 250 nM anti-STAT3 VHH13 data point was omitted as itis an outlier. It was determined that the anti-STAT3 VHH13 (SEQ ID NO.3) sdAb affinity was 1.16×10⁻⁷. The affinity of anti-KRAS VHH was notdetermined.

TABLE 1 Local fit analysis, highlighted values used to determine theaffinity to be 1.16 × 10⁻⁷ VHH Conc. Sensor Type Sample ID LoadingSample ID (nM) KD (M) kon (1/Mb) koff (1/s) Full R{circumflex over ( )}2APS (Aminopropylsilane) ANTI-STAT3 VHH13 STAT3 20 μg/ml 1000 1.168E−073.16E+05 3.69E−02 0.985 APS (Aminopropylsilane) ANTI-STAT3 VHH13 STAT320 μg/ml 500 1.012E−07 4.04E+05 4.09E−02 0.974 APS (Aminopropylsilane)ANTI-STAT3 VHH13 STAT3 20 μg/ml 250  <1.0E−12 4.69E+91 5.11E−02 0.980APS (Aminopropylsilane) ANTI-STAT3 VHH13 STAT3 20 μg/ml 125 1.474E−073.09E+05 4.55E−02 0.991 APS (Aminopropylsilane) ANTI-STAT3 VHH13 STAT320 μg/ml 62.5 9.921E−08 2.71E+05 2.69E−02 0.975 APS (Aminopropylsilane)ANTI-STAT3 VHH13 STAT3 20 μg/ml 31.3  1.53E−06 6.75E+04 1.03E−01 0.656APS (Aminopropylsilane) ANTI-kras STAT3 20 μg/ml 1000  6.75E−08 1.19E+048.01E−04 0.917 APS (Aminopropylsilane) ANTI-kras STAT3 20 μg/ml 5002.916E−08 1.65E+04 4.80E−04 0.890 APS (Aminopropylsilane) ANTI-krasSTAT3 20 μg/ml 250 4.324E−09 8.93E+04 3.86E−04 0.276 APS(Aminopropylsilane) ANTI-kras STAT3 20 μg/ml 125 NA NA NA NA APS(Aminopropylsilane) ANTI-kras STAT3 20 μg/ml 62.5 NA NA NA NA APS(Aminopropylsilane) ANTI-kras STAT3 20 μg/ml 31.3 NA NA NA NA

Example 2 Immunoprecipitation Studies

The specificity of STAT3 sdAbs was assayed in human breast cancer cells.In this example, MDA-MB-231 human breast cancer cells were grown to 50%to 70% confluence. The cells were then disrupted in freshly preparedice-cold lysis buffer (20 mM HEPES, pH 7.9, 400 mM NaCl, 0.1% NP-40, 10%glycerol, 1 mM sodium vanadate, 1 mM sodium fluoride, 1 mMdithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 10 μg/mL aprotinin,10 μg/mL leupeptin) for 45 minutes on ice. Lysates were thencentrifuged, the supernatant collected, and protein concentration wasdetermined using a modified Lowry method (Bio Rad, Hercules, Calif.).Total protein (1 mg) was incubated with 1.5 mg of Dynabeads (Invitrogen)with sdAbs against STAT3, a positive control (STAT3, cat#SC-482, SantaCruz Biotechnology, Dallas, Tex.), or negative control (STAT-1,cat#9172, Cell Signaling, Danvers, Mass.) for 1 hr at 4° C. Beads werethen washed. Following the final wash, 60 μl of lysis buffer was added,and the resulting supernatant was subject to Western blot analysis.Briefly, samples were separated on 10% polyacrylamide gels andtransferred to a nitrocellulose membrane. The membranes were blocked,then incubated with appropriate primary and secondary antibodies.Anti-STAT3 antibody, used as a positive control, was from Cell Signaling(Cat#4904, Danvers, Mass.). The chemiluminescence reaction was performedusing the ECL system from Santa Cruz Biotechnology (Dallas, Tex.).

As illustrated in FIG. 3, endogenous STAT3 immunoprecipitated with allsdAbs tested at varying amounts. M is the Marker lane containing themarker, lane 1 contained STAT3 VHH13 (SEQ ID NO:3) produced and isolatedfrom mammalian cells, lane 2 contained STAT3 VHH14 (SEQ ID NO:4)produced and isolated from mammalian cells, lane 3 contained STAT3 VHH13(SEQ ID NO:3) produced and isolated from bacterial cells, lane 4contained STAT3 VHH14 (SEQ ID NO:4) produced and isolated from mammaliancells, lane 5 was the positive STAT3 antibody, lane 6, used STAT-1 as anegative control, showed no band.

Example 3 Anti-STAT3 Bacterial VHH13 Binds with High Affinity to CellLines Containing Constitutively Actived STAT3

The specificity of bacterial anti-STAT3 VHH13 (SEQ ID NO:3) usingconstitutively activated STAT3 in human (PANC-1 and DU145) and murine(4T1) cell lines was assayed. Commercial HeLa cells were also treatedwith interferon gamma (INFF) in order to induce phosphorylated STAT3.The PC-3 STAT3 null cell line was used as a negative control.

The cells were grown to 50% to 70% confluence, then disrupted in freshlyprepared ice-cold lysis buffer as described above for 45 minutes on ice.Lysates were then centrifuged, the supernatant collected, and proteinconcentration was determined as described above. Total protein (1 mg)was incubated with 1.5 mg of Dynabeads (Invitrogen) containing thebacterial anti-STAT3 VHH13 (SEQ ID NO:3) or negative control (KRAS,Creative Biolabs, Shirley, N.Y.) for 1 hour at 4° C. Beads were thenwashed. Following the final wash, 60 μl of lysis buffer was added, andthe resulting supernatant was subject to Western Blot analysis asdescribed in Example 2.

As illustrated in FIG. 4, endogenous STAT3 was immunoprecipitated bybacterial VHH13 STAT3 (SEQ ID NO:3) in the constitutively activatedSTAT3 cell lines: PANC-1 (lane 1), DU145 (lane 2), and 4T1 (lane 4).Furthermore, bacterial VHH13 STAT3 (SEQ ID NO:3) bound to thePhospho-STAT3 in HeLa lysate (lane 3). No bands were noted for eitherPANC-1 KRAS, lane 3, and PC-3 (negative control), lane 6.

Example 4 Cytotoxicity Studies of Anti-STAT3 sdAbs in MDA-MB-231 CancerCell Lines

In this example, the anti-proliferative effects of anti-STAT3 sdAbs wereassayed using the human breast cancer cell line MDA-MB-231. For theexperiments, MDA-MB-231 cells were grown until they reached a confluencyof 90%. At that time, cells were washed, trypsinized and counted using aCoulter Counter (Beckman, Brea, Calif.). The proliferation studies werecarried out using the 3-[4,5-dimethylthiaolyl]-2,5-diphenyltetrazoliumbromide (MTT) assay. For this, cells were seeded in a 96-well plate at adensity of 5×10³ per well as indicated by the manufacturer (RocheDiagnostics Corporation, Indianapolis, Ind.). Cells were allowed toadhere for 24 hours and then the sdAbs were added at the appropriateconcentrations (i.e., 0, 0.5, 1.0, 10.0, or 100 μg/ml). Cells werecounted on day 3. For the 5-day treated cells, fresh media containingthe sdAbs was refreshed on day 3. At the time of termination, 10 μl ofMTT reagent (0.5 mg/mL) was added to each well as indicated by themanufacturer. After a 4 hour incubation period, 100 μl of solubilizationsolution was added and the plate was placed in the incubator overnight.All the plates were read at 570 nm wavelength using the Biotek platereader (Winooski, Vt.).

All data were analyzed using GraphPad InStat 3 (GraphPad Software, Inc.,La Jolla, Calif.). Treatments groups were compared with vehicle controlgroup using one-way ANOVA. If a significant difference (p<0.05) wasobserved, the Tukey-Kramer multiple comparison test was conducted.

Based on the MTT experiment, the bacterial VHH13 anti-STAT3 (SEQ ID NO.3) sdAb was found to be effective in inhibiting cell growth at days 3and 5 post-treatment, as shown in Tables 2-5 below.

TABLE 2 Mean Absorbance (570 nM) ± S.E. Day 3 Post Treatment withAnti-STAT3 sdAbs in MDA-MB-231 Cells Treatment Control 0.5 μg/ml 1.0μg/ml 10.0 μg/ml 100 μg/ml p-value* H.VHH13 0.444 ± 0.030 0.504 ± 0.0430.545 ± 0.060 0.603 ± 0.025 0.272 ± 0.011 0.001 H.VHH14 0.404 ± 0.0110.485 ± 0.040 0.402 ± 0.017 0.588 ± 0.020 0.416 ± 0.030 0.002 B.VHH130.550 ± 0.036 0.685 ± 0.018 0.716 ± 0.023 0.355 ± 0.033 0.059 ± 0.001<0.0001 B.VHH14 0.593 ± 0.014 0.666 ± 0.022 0.644 ± 0.045 0.456 ± 0.0480.255 ± 0.005 <0.0001 *One Way Analysis of Variance (ANOVA);Tukey-Kramer Multiple Comparison Test

TABLE 3 Effects of Anti-STAT3 sdAb Treatment on MDA-MB-231 CellProliferation after 3 Days of Treatment Treatment μg/ml % Inhibitionp-value* H.VHH13 0.5 NS 1.0 NS 10.0 NS 100.0 38.7 P < 0.05 H.VHH14 0.5NS 1.0 0.5 NS 10.0 NS 100.0 NS B.VHH13 0.5 NS 1.0 NS 10.0 35.5 P < 0.001100.0 89.3 P < 0.001 B.VHH14 0.5 NS 1.0 NS 10.0 23.1 P < 0.05 100.0 57.0P < 0.001 *One Way Analysis of Variance (ANOVA); Tukey-Kramer MultipleComparison Test

TABLE 4 Mean Absorbance (570 nM) ± S.E. Day 5 Post Treatment withAnti-STAT3 sdAb in MDA-MB-231 Cells Treatment Control 0.5 μg/ml 1.0μg/ml 10.0 μg/ml 100 μg/ml p-value* H.VHH13 1.100 ± 0.088 0.955 ± 0.0130.963 ± 0.018 0.832 ± 0.028 0.721 ± 0.025 0.0012 H.VHH14 0.983 ± 0.0230.890 ± 0.021 0.935 ± 0.037 0.804 ± 0.015 0.797 ± 0.010 0.0007 B.VHH130.804 ± 0.046 0.761 ± 0.055 0.653 ± 0.024 0.506 ± 0.030 0.083 ± 0.005<0.0001 B.VHH14 0.677 ± 0.015 0.733 ± 0.038 0.794 ± 0.023 0.640 ± 0.0110.549 ± 0.023 <0.0001 *One Way Analysis of Variance (ANOVA);Tukey-Kramer Multiple Comparison Test

TABLE 5 Effects of Anti-STAT3 sdAb Treatment on MDA-MB-231 CellProliferation After 5 Days of Treatment Treatment μg/ml % Inhibitionp-value* H.VHH13 0.5 13.2 NS 1.0 12.5 NS 10.0 24.4 P < 0.01 100.0 34.5 P< 0.001 H.VHH14 0.5 9.5 NS 1.0 4.9 NS 10.0 18.2 P < 0.001 100.0 18.9 P <0.001 B.VHH13 0.5 5.4 NS 1.0 18.8 NS 10.0 37.1 P < 0.001 100.0 89.7 P <0.001 B.VHH14 0.5 0 NS 1.0 0 NS 10.0 5.5 NS 100.0 18.9 P < 0.05 *One WayAnalysis of Variance (ANOVA); Tukey-Kramer Multiple Comparison Test

Example 5 Cytotoxicity Studies of Anti-STAT3 sdAbs in Human Breast(MDA-MB-231) and Pancreatic (PANC-1) Cancer Cell Lines

In this Example, the anti-proliferative effects of anti-STAT3 VHH13 (SEQID NO. 3) and the VHH14 (SEQ ID NO. 4) sdAbs were assayed using thehuman breast cancer cell line MDA-MB-231 and the human pancreatic cancercell line PANC-1. For the experiments, MDA-MB-231 and PANC-1 cells weregrown until they were 90% confluent. At that time, cells were washed,trypsinized and counted using a Coulter Counter (Beckman, Brea, Calif.).The proliferation studies were carried out using the MTT assay describedabove. For the 5-day treated cells, fresh media containing theanti-STAT3 sdAbs was refreshed on day 3.

All data were analyzed using GraphPad InStat 3. Treatments groups werecompared with vehicle control group using one-way ANOVA. If asignificant difference (p<0.05) was observed, the Tukey-Kramer multiplecomparison test was conducted.

Based on the MTT experiment, both the VHH13 (SEQ ID NO. 3) and the VHH14(SEQ ID NO. 4) were found to inhibit cell growth in both the MDA-MB-231and PANC-1 cancer cells, as shown in Tables 6-13 below.

TABLE 6 Mean Absorbance (570 nM) ± S.E. Day 3 Post Treatment With sdAbsin the MDA-MB-231 Cells Treatment Experiment Control 10.0 μg/ml 100μg/ml p-value* B.VHH13 1 0.550 ± 0.036 0.355 ± 0.033 0.059 ± 0.001<0.0001 2 0.735 ± 0.092 0.489 ± 0.019 0.449 ± 0.054 0.0355 3 0.627 ±0.033 0.432 ± 0.060 0.078 ± 0.001 0.0002 4 0.648 ± 0.090 0.576 ± 0.0610.063 ± 0.002 0.0011 Overall Mean 0.640 ± 0.038 0.463 ± 0.047 0.163 ±0.10  0.0019 B.VHH14 1 0.593 ± 0.014 0.456 ± 0.048 0.255 ± 0.005 0.00052 0.624 ± 0.046 0.499 ± 0.018 0.357 ± 0.019 0.0025 3 0.816 ± 0.088 0.502± 0.048 0.308 ± 0.021 0.0026 4 0.729 ± 0.051 0.559 ± 0.041 0.287 ± 0.0210.0007 Overall Mean 0.691 ± 0.051 0.504 ± 0.021 0.302 ± 0.043 <0.0001*One Way Analysis of Variance (ANOVA); Tukey-Kramer Multiple ComparisonTest

TABLE 7 Mean Absorbance (570 nM) ± S.E. Day 5 Post Treatment withAnti-STAT3 sdAbs in MDA-MB-231 Cells Treatment Experiment Control 10.0μg/ml 100 μg/ml p-value* B.VHH13 1 0.804 ± 0.046 0.506 ± 0.030 0.083 ±0.005 <0.0001 2 0.561 ± 0.024 0.417 ± 0.011 0.266 ± 0.015 <0.0001 30.970 ± 0.048 0.814 ± 0.052 0.105 ± 0.005 <0.0001 4 0.757 ± 0.118 0.665± 0.036 0.087 ± 0.004 0.011 Overall Mean 0.773 ± 0.084 0.601 ± 0.0880.135 ± 0.044 0.0005 B.VHH14 1 0.677 ± 0.015 0.640 ± 0.011 0.549 ± 0.0230.0047 2 0.456 ± 0.037 0.338 ± 0.023 0.274 ± 0.032 0.0166 3 0.983 ±0.019 0.930 ± 0.044 0.578 ± 0.039 0.0004 4 1.092 ± 0.053 0.842 ± 0.0520.499 ± 0.036 0.0004 Overall Mean 0.802 ± 0.145 0.688 ± 0.131  0.475 ±0.0690 0.2022 *One Way Analysis of Variance (ANOVA); Tukey-KramerMultiple Comparison Test

TABLE 8 Mean Absorbance (570 nM) ± S.E. Day 3 Post Treatment withAnti-STAT3 sdAbs in the PANC-1 Cells Treatment Experiment Control 10.0μg/ml 100 μg/ml p-value* B.VHH13 1 0.756 ± 0.045 0.432 ± 0.015 0.307 ±0.012 <0.0001 2 1.347 ± 0.189 0.491 ± 0.087 0.169 ± 0.094 0.0019 3 1.025± 0.056 0.493 ± 0.029 0.166 ± 0.028 <0.0001 Overall Mean 1.043 ± 0.1710.472 ± 0.020 0.214 ± 0.047 0.0034 H.VHH13 1 1.541 ± 0.097 1.066 ± 0.1530.732 ± 0.015 0.0046 2 1.611 ± 0.119 1.353 ± 0.119 0.762 ± 0.654 0.35273 1.074 ± 0.040 0.897 ± 0.154 0.700 ± 0.082 0.1092 Overall Mean 1.409 ±0.169 1.105 ± 0.133 0.731 ± 0.181 0.0238 H.VHH14 1 1.195 ± 0.205 0.920 ±0.133 0.808 ± 0.239 0.4161 2 1.423 ± 0.038 1.183 ± 0.114 0.993 ± 0.0880.0338 3 1.293 ± 0.169 1.163 ± 0.044 0.916 ± 0.088 0.1330 Overall Mean1.304 ± 0.066 1.089 ± 0.085 0.906 ± 0.054 0.0188 *One Way Analysis ofVariance (ANOVA); Tukey-Kramer Multiple Comparison Test

TABLE 9 Mean Absorbance (570 nM) ± S.E. Day 5 Post Treatment withAnti-STAT3 sdAbs in PANC-1 Cells Treatment Experiment Control 10.0 μg/ml100 μg/ml p-value* B.VHH13 1 0.687 ± 0.047 0.433 ± 0.036 0.243 ± 0.0240.0004 2 1.670 ± 0.196 0.869 ± 0.053 0.211 ± 0.006 0.0004 3 1.389 ±0.044 0.627 ± 0.073 0.203 ± 0.013 <0.0001 Overall Mean 1.249 ± 0.2920.643 ± 0.126 0.219 ± 0.012 0.0208 H.VHH13 1 1.462 ± 0.150 1.128 ± 0.1050.839 ± 0.117 0.0349 2 1.792 ± 0.202 1.341 ± 0.095 0.911 ± 0.079 0.01133 1.605 ± 0.289 1.161 ± 0.140 0.820 ± 0.005 0.0638 Overall Mean 1.620 ±0.096 1.210 ± 0.066 0.857 ± 0.028 0.0007 H.VHH14 1 1.992 ± 0.105 1.859 ±0.033 0.095 ± 0.003 <0.0001 2 1.517 ± 0.050 1.165 ± 0.015 1.169 ± 0.0500.0015 3 1.579 ± 0.134 1.081 ± 0.103 0.998 ± 0.049 0.0136 Overall Mean1.696 ± 0.149 1.368 ± 0.247 0.754 ± 0.333 0.0967 *One Way Analysis ofVariance (ANOVA); Tukey-Kramer Multiple Comparison Test

TABLE 10 Mean Growth Inhibition Post 3 Days of Anti-STAT3 sdAbsTreatment on MDA-MB-231 Cell Proliferation Treatment ExperimentP-value^(a) 10.0 μg/ml P-value^(b) 100 μg/ml P-value^(b) B.VHH13 1 P <0.0001 35.5 P < 0.001 89.3 P < 0.001 2 P = 0.03 33.5 ns 38.9 P < 0.05 3P = 0.0001 31.1 P < 0.05 87.6 P < 0.001 4 P = 0.0001 11.1 ns 90.3 P <0.01 Overall 27.8 76.5 Average % Inhibition B.VHH14 1 P < 0.001 23.1 P <0.05 57.0 P < 0.001 2 P = 0.03 20.0 ns 42.8 P < 0.01 3 P = 0.03 38.5 P <0.05 62.3 P < 0.01 4 P = 0.006 23.3 ns 60.6 P < 0.001 Overall 26.2 55.7Average % Inhibition ^(a)One-way Analysis of Variance (ANOVA); ^(b)Posttest = Tukey-Kramer Multiple Comparisons Test

TABLE 11 Mean Growth Inhibition Post 5 days of Anti-STAT3 sdAbsTreatment on MDA-MB-231 Cell Proliferation Treatment ExperimentP-value^(a) 10.0 μg/ml P-value^(b) 100 μg/ml P-value^(b) B.VHH13 1 P <0.0001 37.1 P < 0.001 89.7 P < 0.001 2 P < 0.0001 25.7 P < 0.001 52.6 P< 0.001 3 P < 0.0001 16.1 ns 89.2 P < 0.001 4 P = 0.001 12.2 ns 88.5 P <0.01 Overall 22.8 80.0 Average % Inhibition B.VHH14 1 P < 0.0001 5.5 ns18.9 P < 0.05 2 P = 0.02 25.9 ns 39.9 P < 0.05 3 P = 0.0004 5.4 ns 41.2P < 0.001 4 P = 0.0004 22.9 P < 0.05 54.3 P < 0.001 Overall 14.9 38.6Average % Inhibition ^(a)One-way Analysis of Variance (ANOVA); ^(b)Posttest = Tukey-Kramer Multiple Comparisons Test

TABLE 12 Mean Growth Inhibition Post 3 Days of Anti-STAT3 sdAbsTreatment on PANC-1 Cell Proliferation Treatment Experiment P-value^(a)10.0 μg/ml P-value^(b) 100 μg/ml P-value^(b) B.VHH13 1 P < 0.0001 42.9 P< 0.001 59.4 P < 0.001 2 P = 0.03 63.5 P < 0.05 87.5 P < 0.01 3 P <0.0001 51.9 P < 0.001 83.8 P < 0.001 Overall 52.8 76.9 Average %Inhibition H.VHH13 1 P = 0.005 30.8 P < 0.05 52.5 P < 0.01 2 P = 0.00216.0 ns 52.7 P < 0.01 3 P = 0.11 16.5 ns 34.8 ns Overall 21.1 46.7Average % Inhibition H.VHH14 1 P = 0.42 23.0 ns 32.4 ns 2 P = 0.03 16.9ns 30.2 P < 0.05 3 P = 0.13 10.1 ns 29.2 ns Overall 16.7 30.6 Average %Inhibition ^(a)One-way Analysis of Variance (ANOVA); ^(b)Post test =Tukey-Kramer Multiple Comparisons Test

TABLE 13 Mean Growth Inhibition Post 5 Days of Anti-STAT3 sdAbsTreatment on PANC-1 Cell Proliferation Treatment Experiment P-value^(a)10.0 μg/ml P-value^(b) 100 μg/ml P-value^(b) B.VHH13 1 P = 0.0004 37.0 P< 0.01 64.6 P < 0.001 2 P = 0.0004 48.0 P < 0.01 87.4 P < 0.001 3 P <0.0001 54.9 P < 0.001 85.4 P < 0.001 Overall 46.6 79.1 Average %Inhibition H.VHH13 1 P = 0.03 22.8 ns 42.6 P < 0.05 2 P = 0.01 25.2 ns49.2 P < 0.01 3 P = 0.06 27.7 ns 48.9 ns Overall 25.2 46.9 Average %Inhibition H.VHH14 1 P = 0.08 26.8 ns 14.8 ns 2 P = 0.002 23.2 P < 0.0122.9 P < 0.01 3 P = 0.02 31.5 P < 0.05 36.8 P < 0.05 Overall 27.2 24.8Average % Inhibition ^(a)One-way Analysis of Variance (ANOVA); ^(b)Posttest = Tukey-Kramer Multiple Comparisons Test

Example 6 Anti-Proliferative Actions of STAT3 sdAbs in the Human BreastCancer and Human Prostate Cancer Cell Lines

The anti-proliferative effects of the STAT3 VHH13 (SEQ ID NO. 3) sdAbwere assayed in the human breast cancer cell line MDA-MB-231 and thehuman prostate cancer cell lines DU145. For the experiments, cancercells were grown until they reached 90% confluence. At that time, cellswere washed, trypsinized, and counted using a Coulter Counter (Beckman,Brea, Calif.). The proliferation studies done using the MTT assay asdescribed above.

The anti-proliferative properties of anti-STAT3 bacterial VHH13 (SEQ IDNO. 3) sdAb on MDA-MB-231 cells were compared to its actions on DU145cells. As shown in Table 14, MDA-MB-231 cells treated with theanti-STAT3 (SEQ ID NO:3) sdAbs showed an average growth inhibition of29.6 and 91.2 at 50.0 and 100 μg/ml, respectively. In the DU145 cells, asimilar growth inhibition (31.2 and 92.1% for 50.0 and 100 μg/ml,respectively) was seen as set forth in Table 15.

TABLE 14 Anti-proliferative Actions of Anti-STAT3 Bacterial VHH13 sdAbson MDA-MB-231 Breast Cancer Cells Experiment 1 Experiment 2 Experiment 3Average Absorbance Absorbance Absorbance Absorbance (% Inhibition) (%Inhibition) (% Inhibition) (% Inhibition) p-value* control 0.93 1.251.46 1.21  50 μg 0.82 (12.0) 0.99 (20.5) 0.64 (56.2) 0.82 (32.6) NS 100μg 0.07 (93.1) 0.12 (90.1) 0.14 (90.5) 0.11 (91.0) <0.001 *One WayAnalysis of Variance (ANOVA); Tukey-Kramer Multiple Comparison Test

TABLE 15 Anti-proliferative Actions of Anti-STAT3 Bacterial VHH13 sdAbson DU145 Prostate Cancer Cells Experiment 1 Experiment 2 Experiment 3Average Absorbance Absorbance Absorbance Absorbance (% Inhibition) (%Inhibition) (% Inhibition) (% Inhibition) p-value* control 1.05 1.581.61 1.41 50 μg 0.68 (35.7)  1.2 (55.5) 1.03 (35.8) 0.98 (30.5) NS 100μg 0.13 (87.4) 0.12 (95.7) 0.06 (96.1) 0.10 (92.7) <0.001 *One WayAnalysis of Variance (ANOVA); Tukey-Kramer Multiple Comparison Test

Example 7 Anti-Proliferative Effects of STAT3 VHH13 (SEQ ID NO. 3) sdAbson Human Cancer Cell Lines

To test the anti-proliferative effects of the STAT3 VHH13 (SEQ ID NO. 3)sdAbs using the human cancer cell lines: MDA-MB-231, MDA-MB-468, MCF-7,BT474, and DU145 as shown in Table 16.

All human cancer cell lines were obtained from American Type CultureCollection (Manassas, Va.). Cell lines were maintained and cultured inRPMI 1640 media (MDA-MB-231, MDA-MB-468, MCF-7, BT474) or MEM-E (DU145)containing 10% fetal bovine serum, 2 mM L-glutamine and 1%antibiotic-antimycotic solution (10 units/mL penicillin, 10 μg/mLstreptomycin and 25 μg/mL amphotericin B). Cells were kept at 37° C. ina humidified atmosphere of 5% CO₂. Cell culture supplies were obtainedfrom Life Technologies, Inc., (Grand Island, N.Y.). The MTT reagent waspurchased from Sigma Aldrich (St. Louis, Mo.).

For the experiments, cancer cells were grown until they reached 90%confluency. At that time, cells were washed, trypsinized and countedusing a Coulter Counter (Beckman, Brea, Calif.). The proliferationstudies were carried out using the MTT assay as described above.

The anti-proliferative properties of Anti-STAT3 Bacterial VHH13 (SEQ IDNO:3) sdAbs were evaluated on five breast cancer cells of representingvarious classifications (Table 34). As shown in Table 17, all cell linesat 72 hours post treatment showed significant growth inhibition. Thegreatest growth inhibition was noted at 100 and 200 μg/ml dose for allcell lines. The half maximal inhibitory concentration (IC₅₀) for growthin the cell lines tested were: 10.1±2.4, 12.36±1.5, 14.8±1.6, and25.2±14.7 for the MDA-MB-231, MDA-MB-468, MCF-7, and BT474 cell lines,respectively. These data suggest that the triple negative breast cancercell lines require the lowest concentration of VHH13 (SEQ ID NO:3) sdAbsto achieve the IC₅₀ as compared to estrogen/progesterone positive celllines (i.e., MCF-7) or HER2 amplified cell lines (i.e., BT474).

TABLE 16 Breast Cancer Cell Line Characteristics Cell line DiseaseImmunoprofile Classification MDA-MB-231 adenocarcinoma ER⁻, PR⁻, HER2⁻Basal; Claudin-low MDA-MB-468 adenocarcinoma ER⁻, PR⁻, Her2⁻ BasalMDA-MB-453 metastatic ER, PR, HER2⁻ Unclassified carcinoma BT474 ductalcarcinoma Her2 amplified Luminal B MCF-7 adenocarcinoma ER⁺, PR⁺, HER2⁺Luminal A

TABLE 17 Inhibition of Breast Cancer Cell Lines by Anti-STAT3 VHH13 (SEQID NO. 3) sdAbs Cell Line Treatment (μg/ml) Mean Abs % Inhibitionp-value BT474 0 0.634 0.39 0.322 49.3 P < 0.001 0.78 0.462 27.2 P <0.001 1.56 0.502 20.8 P < 0.01 3.13 0.446 29.7 P < 0.001 6.25 0.469 26.1P < 0.001 12.5 0.363 42.7 P < 0.001 25 0.256 59.6 P < 0.001 50 0.14577.2 P < 0.001 100 0.046 92.8 P < 0.001 200 0.040 93.8 P < 0.001 MCF-7 00.590 0.39 0.818 0 0.78 0.785 0 1.56 0.823 0 3.13 0.689 0 6.25 0.43522.1 NS 12.5 0.327 41.6 P < 0.01 25 0.212 62.1 P < 0.001 50 0.057 89.9 P< 0.001 100 0.038 93.2 P < 0.001 200 0.040 92.9 P < 0.001 MDA-MB-468 00.253 0.39 0.311 0 0.78 0.289 0 1.56 0.201 20.6 3.13 0.223 11.9 6.250.230 9.1 12.5 0.130 48.6 P < 0.001 25 0.067 73.5 P < 0.001 50 0.04283.4 P < 0.001 100 0.038 85.0 P < 0.001 200 0.040 84.4 P < 0.001MDA-MB-231 0 0.502 0.39 0.603 0 0.78 0.576 0 1.56 0.570 0 3.13 0.44511.4 P < 0.001 6.25 0.312 37.8 P < 0.001 12.5 0.224 55.4 P < 0.001 250.196 60.9 P < 0.001 50 0.130 74.2 P < 0.001 100 0.041 91.8 P < 0.001200 0.042 91.7 P < 0.001

The actions of anti-STAT3 bacterial VHH13 (SEQ ID NO:3) sdAb was alsoevaluated in the human prostate cancer cell line DU145, as shown inTable 18. The anti-STAT3 bacterial VHH13 (SEQ ID NO:3) sdAb showeddose-dependent growth inhibition in all cancer cells tested.

TABLE 18 Effect of Anti-STAT3 VHH13 sdAbs on Prostate Cancer Cell LinesTreatment (mg/ml) Mean Abs % Inhibition p-value DU145 0 0.771 DU145 0.390.906 0 DU145 0.78 1.023 0 DU145 1.56 0.967 0 DU145 3.13 0.783 0 DU1456.25 0.770 0 DU145 12.5 0.560 27.4 P < 0.05 DU145 25 0.359 53.5 P <0.001 DU145 50 0.161 79.1 P < 0.001 DU145 100 0.039 95.0 P < 0.001 DU145200 0.039 95.0 P < 0.001

Example 8 Maximum Tolerated Dose of Anti-STAT3 Bacterial VHH13 (SEQ IDNO:3) in BALB/C Mice

In this Example, the tolerance of anti-STAT3 bacterial VHH13 (SEQ IDNO:3) sdAb was assayed in test animals using the human breast cancercell line MDA-MB-231. For the experiment, a total of 9 BALB/C nudefemale mice (6 to 7 weeks old) were divided into three groups accordingto body weights. (Table 19) Mice (n=3) received either vehicle (PBS) oranti-STAT3 bacterial VHH13 (SEQ ID NO:3) sdAb at 250 or 500 μg/kg bodyweight/day for five days. During the study, mortality/morbidity wasperformed twice daily. Body weights were recorded on days 1, 4, and 6 ofthe study as well as on the day of study termination (Day 13). Toxicitywas assessed by body weight measurements and mouse behavior compared tovehicle control mice. Upon completion of treatment phase, animals werefollowed for an additional week to note any abnormalities in bodyweights and/or general health post treatment.

TABLE 19 Experimental Design of Maximum Tolerated Dose Study Group #Mice Treatment Dose Route Frequency 1 3 PBS Vehicle — IP 5 days 2 3Bacterial VHH13 250 μg/kg b.w. IP 5 days 3 3 Bacterial VHH13 500 μg/kgb.w. IP 5 days

As illustrated in Table 20, there was no significant difference in bodyweights among the groups, and anti-STAT3 bacterial VHH13 (SEQ ID NO:3)sdAb was not associated with any drug-related deaths at either dosinglevel. Additionally, no behavior changes were observed in the animalstreated with anti-STAT3 bacterial VHH13 (SEQ ID NO:3) sdAb as comparedto the control mice.

TABLE 20 Mean body weights ± S.E Group Randomization Day 1 Day 4 Day 6Day 13 Vehicle 17.1 ± 0.06 17.1 ± 0.07 17.8 ± 0.12 18.1 ± 0.09 18.8 ±0.20 250 μg/kg 17.1 ± 0.06 17.2 ± 0.03 17.2 ± 0.15 17.5 ± 0.15 18.1 ±0.21 500 μg/kg 17.1 ± 0.17 17.1 ± 0.09 17.8 ± 0.18 18.0 ± 0.20 18.5 ±0.18 p-value* >0.9999 0.52 0.05 0.07 0.11 *One Way Analysis of Variance(ANOVA); Tukey-Kramer Multiple Comparison Test

Example 9 Activity of Bacterial Anti-STAT3 VHH13 (SEQ ID NO:3) in NudeBALB/C Mice Xenograft and Human Breast Cancer and Human PancreaticCancer Cells

In this example, the activity of anti-STAT3 bacterial VHH13 (SEQ IDNO:3) sdAb was evaluated in mice using the human breast cancer cell lineMDA-MB-231. Briefly, the activity of anti-STAT3 bacterial VHH13 (SEQ IDNO:3) sdAb was evaluated using: the MDA-MB-231 human breast cancerxenograft model and the PANC-1 human pancreatic cancer xenograft model.Dosing schedules were as follows: Group 1 (n=6; PBS; IP) daily for 14days [QD×14]; and Group 2 (n-12; 500 μg/kg bw; IP), every day for 14days [QD×14]. An observation period of 5 days followed the drugadministration.

The human breast cancer cell lines (MDA-MB-231 and PANC-1) were obtainedfrom American Type Culture Collection (ATCC) (Manassas, Va.). TheMDA-MB-231 cells were growth in MEM (Life Technologies, Grand Island,N.Y.) supplemented with 10% FBS (Atlanta Biologicals, Flowery Branch,GA) and Penicillin-Streptomycin-Glutamine (Life Technologies, GrandIsland, N.Y.). The PANC-1 cells were grown in RPMI 1640 media (LifeTechnologies, Grand Island, N.Y.) supplemented with 10% FBS andPenicillin-Streptomycin-Glutamine. All cells were grown in the presenceof 5% CO₂ at 37° C. in an incubator.

Athymic nude-Foxn1^(nu) male mice aged 4 to 5 weeks were purchased fromHarlan Laboratories (Indianapolis, Ind.). Animals were quarantined forone week and housed five mice per cage, with a 12-hr light-dark cycle,and a relative humidity of 50%. Drinking water and diet were supplied tothe animals ad libitum. All animals were housed under pathogen-freeconditions and experiments were performed in accordance with the IITResearch Institute Animal Use and Care Committee. For the MDA-MB-231xenograft study, cells (4×10⁶) in a 100-μL final volume of MEM mediawere injected subcutaneously into right flanks of mice. For the PANC-1xenograft study, cells (5×10⁶) in a 100-μL final volume of RPMI mediawere injected subcutaneously into right flanks of mice. Tumormeasurements for both models were initiated as soon as the tumors werepalpable. Thereafter, tumors were measured twice weekly. Animals wererandomized when tumors reach a range size of 75 to 175 mm³, control(n=6) and a treatment (n=12) groups were randomized using the stratifiedrandom sampling algorithm. Treatment (anti-STAT3 bacterial VHH13 (SEQ IDNO:3) sdAb) or Vehicle (PBS) was initiated the day followingrandomization. The treatment was well tolerated and associated with nodrug-related deaths. No significant body weight loss was noted.

For the MDA-MB-231 xenograft study, the randomization Mean (±SE) tumorsize was: 103.01±11.89 and 102.61±9.60 for control and treatment groupsrespectively. Mean body weights (±SE) at randomization were: 32.08±0.76and 30.27+0.75 for Group 1 and Group 2, respectively. Table 21 shows themean body weights (±SE) for the entire study.

TABLE 21 Mean body weights ± S.E. Treatment Day 1 Day 6 Day 9 Day 12 Day16 Day 20 Vehicle 31.0 ± 0.83 32.1 ± 0.76 31.9 ± 0.66 32.1 ± 0.68 32.0 ±0.71 32.5 ± 0.88 Anti-STAT3 29.2 ± 0.71 30.3 ± 0.75 30.4 ± 0.79 29.9 ±0.72 30.6 ± 0.74 30.6 ± 0.77 VHH13 p-value* 0.16 0.18 0.27 .09 0.28 0.17*Two-tail T-Test

On day 14 of dosing, the mean tumor size (±SE) for the control was179.11±19.39 versus 118.86±15.94 for treatment group. Mean body weights(±SE) at termination were: 31.98±0.71 and 30.55±0.74 for Group 1 andGroup 2, respectively. Table 22 summarizes the tumor volumes (±SE) forentire study. The % mean tumor growth inhibition in the treatment groupwas 33.64%. The tumor doubling times were as follows: Group 1: 44.27days; and Group 2: 61.06 days. FIG. 5 illustrates the growth inhibitionof anti-STAT3 bacterial VHH13 (SEQ ID NO:3) sdAb in the MDA-MB-231xenograft model. Anti-STAT3 bacterial VHH13 (SEQ ID NO:3) sdAb showedsignificant growth inhibition (p=0.047). Thus, anti-STAT3 bacterialVHH13 (SEQ ID NO:3) sdAb has chemotherapeutic activity in the MDA-MB-231human breast cancer model system.

TABLE 22 Individual Tumor Measurements (mm³) for the MDA-MB-231Xenograft Model Ani- Group mal # Day 1 Day 6 Day 9 Day 12 Day 16 Day 201 1 117.43 141.72 135.00 139.31 127.93 133.19 2 130.30 142.83 206.15256.99 244.06 243.00 3 78.00 105.97 114.04 144.06 154.50 158.94 4 118.24162.41 171.39 225.59 181.32 217.97 5 71.10 109.03 133.13 168.80 187.73164.45 Mean 103.01 132.39 151.94 186.95 179.11 183.51 S.E. 11.89 10.8216.42 23.28 19.39 20.28 2 6 123.94 114.91 129.22 176.04 170.09 162.98 785.93 101.06 112.60 112.24 139.56 96.43 8 147.34 148.72 169.69 185.08170.07 256.71 9 115.91 103.64 108.37 141.21 144.51 119.42 10 73.23 82.59110.13 91.22 166.77 285.88 11 163.73 178.23 183.79 165.52 214.28 129.5112 75.54 83.94 103.68 119.88 104.26 99.48 13 70.04 89.24 102.60 75.2557.65 95.23 14 101.62 65.09 82.02 68.01 61.41 61.83 15 67.83 62.21 59.0077.04 65.49 82.73 16 131.93 75.28 76.21 53.55 73.66 51.61 17 74.28109.06 111.92 89.94 58.56 100.07 Mean 102.61 101.16 112.44 112.92 118.86128.49 S.E. 9.6 9.8 10.3 12.9 15.9 21.1 P-value 0.98 0.08 0.06 0.01 0.050.14

For the PANC-1 xenograft study, the randomization Mean (+SE) tumor sizeswere 107.01±4.54 in the control and 110.58±6.18 in the treatment groups.Mean body weights (±SE) at randomization were: 29.0±0.81 and 28.5±0.70for Group 1 and Group 2, respectively. Mean body weights (±SE) attermination were: 31.2±0.99 and 30.1±0.75 for Group 1 and Group 2,respectively. Table 23 summarizes the mean body weights (±SE) for entirestudy. On day 14 of dosing, the mean tumor size (±SE) for control was287.30±33.94 versus 318.74+29.76 for treatment group. Table 24summarizes the tumor volumes (±SE) for entire study.

TABLE 23 Mean body weights ± S.E. Treatment 2/19 2/24 2/27 3/2 3/6 3/10Vehicle Control 31.0 ± 0.83 32.1 ± 0.76 31.9 ± 0.66 32.1 ± 0.68 32.0 ±0.71 32.5 ± 0.88 Anti-STAT3 29.2 ± 0.71 30.3 ± 0.75 30.4 ± 0.79 29.9 ±0.72 30.6 ± 0.74 30.6 ± 0.77

The tumor doubling times were as follows: Group 1: 22.44 days; and Group23.02 days. Anti-STAT3 bacterial VHH13 (SEQ ID NO:3) sdAb showed nosignificant growth inhibition in the PANC-1 human pancreatic cancermodel system.

TABLE 24 Individual Tumor Measurements (mm³) for the PANC-1 xenograftModel Ani- Group mal # 2/19 2/24 2/27 3/2 3/6 3/10 1 1 99.77 117.96134.67 161.27 160.79 195.58 2 117.54 137.14 221.14 241.27 303.70 321.453 120.30 210.99 276.05 322.17 394.96 732.07 4 111.65 135.91 215.87340.97 334.08 382.06 5 90.88 96.35 165.26 156.28 223.17 314.97 6 107.05156.56 192.98 324.34 307.13 573.99 Mean 107.87 142.49 201.00 257.72287.30 420.02 S.E. 11.11 16.01 20.00 34.35 33.94 80.34 2 7 96.31 193.71275.06 317.53 395.37 540.66 8 89.24 90.03 112.43 125.51 189.63 235.08 980.62 148.97 196.38 187.24 299.84 530.46 10 108.03 144.14 234.46 240.39288.75 421.61 11 77.66 116.21 313.19 290.38 411.66 197.67 12 129.68143.20 290.67 224.92 261.44 343.04 13 108.99 182.30 239.00 254.64 342.19464.00 14 123.27 171.03 223.34 226.88 248.69 324.30 15 144.53 136.03198.47 226.04 247.97 273.58 16 120.96 136.48 226.43 338.06 564.71 883.8117 112.69 144.76 167.12 225.70 223.06 326.19 18 134.95 189.64 193.14248.01 351.63 364.44 Mean 110.58 149.71 222.47 242.11 318.74 408.74 S.E.6.18 8.79 15.90 16.30 29.76 53.25 P-value 0.78 0.67 0.43 0.64 0.53 0.91

Example 10 MDA-MB-231 Xenograft Study

In this Example, the efficacy of anti-STAT3 bacterial VHH13 (SEQ IDNO:3) sdAb in the MDA-MB-231 human breast xenograft model was furtherevaluated. The dosing schedules were as follows: Group 1 (n=4; PBS; IP)twice a day for 14 days [BID×14]; Group 2 (n=4; 1 mg/kg bw; IP), twice aday for 14 days [BID×14]; Group 3 (n=4; 2 mg/kg bw; IP) twice a day for14 days [BID×14]; and Group 4 (n=4; 2 mg/kg bw; IP) once a day for 14days [QD×14]. An observation period of 7 days followed administration.

The human breast cancer cell lines MDA-MB-231 and athymicnude-Foxn1^(nu) female mice were described above.

MDA-MB-231 cells at a density of 5×10⁶ were injected subcutaneously intothe right flank of the mice at a final volume of 100-μL in MEM media.Tumor measurements were initiated as soon as the tumors were palpable.Thereafter, tumors were measured twice weekly. Animals are randomizedwhen tumors reach a range size of 55 to 150 mm³ using the stratifiedrandom sampling algorithm. Treatment (anti-STAT3 bacterial VHH13 (SEQ IDNO:3) sdAb) or Vehicle (PBS) was initiated the day followingrandomization.

The randomization Mean (±SE) tumor size was: 92.08±13.24, 82.38±5.17,77.47±7.17, and 104.71±14.64 for Groups 1, 2, 3, and 4 respectively. Asshown in Table 25, mean body weights (±SE) at randomization were:23.65±0.72, 23.45±0.66, 23.10±0.20, and 22.45±1.25 for Groups 1, 2, 3,and 4, respectively.

As shown in Table 26, at day 14 of dosing, the mean tumor size (±SE) forcontrol group was 221.51±57.32 versus 67.12±10.66, 58.27±22.54, and131.44±22.86, for treatment group 2, 3, and 4, respectively. At the timeof termination (day 42) mean tumor size (±S.E.) was: 255.42±65.46,55.98±6.94, 41.15±13.21, and 145.51±52.32, for groups 1, 2, 3, and 4,respectively. Mean body weights (±SE) at termination were: 24.80±0.49,23.25±1.20, 24.00±0.32, and 23.2±1.46 for Groups 1, 2, 3, and 4,respectively. The max mean % net weight loss (day) was: 0.7 (36), 1.5(23), 1.8 (36), and 2.2 (29) for Groups 1, 2, 3, and 4, respectively.

Also as shown in Table 26, the mean growth inhibition in the treatmentgroups was 78.3, 75.2, and 55.9, for Groups 2, 3, and 4, respectively.The tumor doubling times were: Group 1: 20.56 days; Group 2: 34.54 days;Group 3: 30.07 days; and Group 4: 27.17 days. There was a growth delayof 13.99, 9.52, and 6.61 days for Groups 2, 3 and 4, respectively. The %treatment/control values for treatment groups were: Group 2: −33.75(tumor stasis); Group 3: −54.4 (tumor regression); and Group 4: 10.28(tumor inhibition). FIG. 6 illustrates the growth inhibition ofanti-STAT3 bacterial VHH13 (SEQ ID NO:3) sdAb in the MDA-MB-231xenograft model.

Administration of anti-STAT3 bacterial VHH13 (SEQ ID NO:3) sdAb wasassociated with a significant growth inhibition in Group 2 (p=0.02) [1mg/kg; BID×14] and Group 3 (p=0.02) [2 mg/kg; BID×14]. Furthermore,three out of four tumors showed significant regression. Based on thesedata, it is concluded that anti-STAT3 bacterial VHH13 (SEQ ID NO:3) sdAbhas chemotherapeutic activity in the MDA-MB-231 human breast cancermodel system.

TABLE 25 Mean Body Weights ± S.E. Date/Study Day Dosing Recovery 6/236/26 6/29 7/2 7/6 7/9 7/15 Group Schedule 20 23 26 29 33 36 42 1 PBS;BID × 14 23.65 ± 0.72 23.85 ± 0.60 24.18 ± 0.67 24.05 ± 0.63 24.30 ±0.67 24.13 ± 0.72 24.80 ± 0.49 2 1 mg/kg; BID × 14 23.45 ± 0.66 23.10 ±0.68 23.13 ± 0.74 23.13 ± 0.95 23.08 ± 1.01 23.13 ± 1.09 23.25 ± 1.20 32 mg/kg; BID × 14 23.10 ± 0.20 23.10 ± 0.14 23.20 ± 0.07 23.85 ± 0.3923.80 ± 0.24 23.38 ± 0.23 24.00 ± 0.32 4 2 mg/kg; QD × 14 22.45 ± 1.2522.35 ± 1.32 22.58 ± 1.46 22.08 ± 1.44 22.73 ± 1.47 22.55 ± 1.46 23.20 ±1.38

TABLE 26 Individual Tumor Measurements (mm³) for the MDA-MB-231Xenograft Model Jun. 23, 2015 Jun. 26, 2015 Jun. 29, 2015 Jul. 2, 2015Jul. 6, 2015 Jul. 9, 2015 Jul. 15, 2015 Animal # (20) (23) (26) (29)(33) (36) (42) Group 1 001 93.38 119.07 159.80 197.91 210.95 243.31265.61 002 116.07 241.31 313.16 339.13 362.30 390.48 426.32 003 55.6783.45 98.22 135.50 198.19 204.96 218.29 004 104.82 112.09 118.44 111.07114.61 115.31 111.45 Mean Absolute 92.49 138.98 172.41 195.90 221.51238.51 255.42 Mean Relative 100.00% 150.27% 186.41% 211.82% 239.51%257.89% 276.17% S.E. Mean 13.12 34.97 48.64 51.12 51.56 57.32 65.46 %Inhibition Mean Median 99.10 115.58 139.12 166.71 204.57 224.13 241.95Absolute Median 100.00% 116.62% 140.38% 168.22% 206.42% 226.16% 244.14%Relative S.E. Median 13.66 37.49 52.30 53.83 52.48 57.91 65.92 %Inhibition Median Group 2 005 73.15 54.54 59.17 57.21 56.20 37.13 39.17006 80.11 76.56 80.34 88.75 99.09 87.42 72.18 007 97.22 79.99 78.4459.90 55.90 53.66 60.35 008 81.21 53.58 54.34 67.43 57.30 29.02 52.23Mean Absolute 82.92 66.17 68.07 68.32 67.12 51.81 55.98 Mean Relative100.00% 79.79% 82.09% 82.39% 80.95% 62.48% 67.51% S.E. Mean 5.09 7.036.62 7.14 10.66 12.93 6.94 % Inhibition 10.34% 52.39% 60.52% 65.12%69.70% 78.28% 78.08% Mean Median 80.66 65.55 68.80 63.66 56.75 45.4056.29 Absolute Median 100.00% 81.27% 85.30% 78.93% 70.36% 56.28% 69.79%Relative S.E. Median 5.25 7.04 6.63 7.63 12.23 13.45 6.94 % Inhibition18.61% 43.28% 50.54% 61.81% 72.26% 79.75% 76.74% Median Group 3 00956.41 43.61 33.13 31.76 34.11 50.33 18.94 010 84.06 85.18 61.75 80.69110.72 89.11 73.89 011 82.87 54.78 34.92 54.38 78.47 78.68 51.30 01286.73 44.01 23.09 16.99 9.78 18.71 20.48 Mean Absolute 77.52 56.89 38.2245.95 58.27 59.21 41.15 Mean Relative 100.00% 73.39% 49.31% 59.28%75.17% 76.38% 53.09% S.E. Mean 7.08 9.78 8.26 13.90 22.54 15.79 13.21 %Inhibition 16.19% 59.06% 77.83% 76.54% 73.69% 75.18% 83.89% Mean Median83.46 49.39 34.02 43.07 56.29 64.51 35.89 Absolute Median 100.00% 59.18%40.76% 51.60% 67.44% 77.29% 43.00% Relative S.E. Median 7.87 10.69 8.6114.00 22.56 16.08 13.56 % Inhibition 15.78% 57.27% 75.54% 74.17% 72.49%71.22% 85.17% Median Group 4 013 88.56 108.35 105.80 102.94 183.39159.78 291.06 014 78.73 51.51 54.20 70.39 84.29 55.83 42.03 015 113.2085.29 69.30 103.16 103.20 87.15 130.64 016 141.91 130.82 87.49 145.68154.89 117.63 118.31 Mean 105.60 93.99 79.20 105.54 131.44 105.10 145.51Absolute Mean Relative 100.00% 89.01% 75.00% 99.94% 124.47% 99.52%137.79% S.E. Mean 14.11 16.94 11.18 15.44 22.86 22.17 52.32 % Inhibition−14.18% 32.37% 54.06% 46.13% 40.66% 55.94% 43.03% Mean Median 100.8896.82 78.40 103.05 129.05 102.39 124.47 Absolute Median 100.00% 95.98%77.71% 102.15% 127.92% 101.49% 123.38% Relative S.E. Median 14.37 17.0211.19 15.50 22.90 22.22 53.72 % Inhibition −1.80% 16.23% 43.65% 38.19%36.92% 54.32% 48.55% Median

Example 11 Efficacy of Anti-STAT3 Bacterial VHH13 (SEQ ID NO:3) sdAb onThree Human Cancer Xenograft Models

In this Example, the efficacy of anti-STAT3 bacterial VHH13 (SEQ IDNO:3) sdAb was evaluated in the MDA-MB-231 Human Breast, PANC-1Pancreatic, and DU145 Prostate cancer xenograft models.

Athymic Nude-Foxn1^(nu) mice, MDA-MB-231 breast cancer cells, PANC-1pancreatic cancer and the DU145 prostate cancer cell lines weredescribed above. The body weight of the mice ranged from 17 to 19 g (34females) and 21 to 23 g (16 males) on Day 1 of the study.

Cells in early passages (4 to 10) were used for implantation into themice and were harvested during log phase growth. MDA-MB-231 (5×10⁶),DU145 (5×10⁶), and PANC-1 (1.5×10⁶) were injected subcutaneously intothe right flank of the mice at a final volume of 100-μL of media. Tumormeasurements were initiated as soon as the tumors were palpable.Thereafter, tumors were measured twice weekly.

Animals were randomized using the stratified random sampling algorithmwhen tumors reach a range size of: 74-120 mm³ (MDA-MB-231), 89-146 mm³(DU145), or 60-160 mm³ (PANC-1). Treatment (containing anti-STAT3bacterial VHH13 (SEQ ID NO:3) sdAb and referred to herein as SBT-100) orVehicle (PBS) was initiated the day following randomization, referred toas day 1.

Anti-STAT3 bacterial VHH13 (SEQ ID NO:3) sdAb was supplied as apre-formulated solution at a concentration of 0.651 mg/ml and was storedat −20° C. until ready to use. The stock solution was diluted in sterilePBS pH 7.6 to provide a 5 mg/kg in a dosing volume of 10 mL/kg. Theworking solution was prepared every 7 days, aliquoted onto seven vialsand stored at 4° C. On each day of treatment, only the needed vial wasbrought to room temperature. All leftover sdAb material was retained at4° C. as need for the next dose. At day 8, any remaining sdAb materialwas discarded and a fresh batch prepared.

Two groups (control and SBT-100) of mice per tumor model were dosedaccording to the protocol shown in Table 27. Dosing schedules were asfollows: Group 1 (n=4; PBS) twice a day for 14 days [BID×14]; Group 2(n=4; SBT-100, 5 mg/kg bw), twice a day for 14 days [BID×14]. Both thevehicle (PBS pH 7.6) and SBT100 were administered intraperitoneally(i.p.) twice a day, six hours apart for fourteen days. Dosing wasconducted according to individual animal weights. A recovery period of 7days followed administration.

TABLE 27 Experimental Design of Xenograft Study # of cells Dose Modelinoculated/mouse Group # Mice Agent (mg/Kg) Route Schedule MDA-MB-231 5× 10⁸ 1 4 Control (PBS) 0 IP BID × 14 2 4 SBT-100 5 IP BID × 14 PANC-11.5 × 10⁸   1 4 Control (PBS) 0 IP BID × 14 2 4 SBT-100 5 IP BID × 14DU145 5 × 10⁸ 1 4 Control (PBS) 0 IP BID × 14 2 4 SBT-100 5 IP BID × 14

Study Log Study Director Animal Study Management Software (SanFrancisco, Calif.) was used to randomize animals, collect data (e.g.,dosing, body weights, tumors measurements, clinical observations), andconduct data analyses.

In the MDA-MB-231 tumor xenograft model, animals were randomized on day23 post-inoculation with a mean (±SE) tumor size of: 77.98±21.58 and84.71±5.56 for Groups 1 and 2, respectively. Mean body weights (±SE) atrandomization were: 20.04±0.62 and 23.7±1.84 for Groups 1 and 2,respectively. Table 28 summarizes the mean body weights (±SE) for entirestudy. At last day of dosing (Day 14), the mean tumor size (±SE) forcontrol group was 168.28±51.57 versus 83.81±22.65 for SBT-100 treatedmice. Table 29 summarizes the tumor volumes (±SE) for entire study. Atthe time of termination (day 28) mean tumor size (±S.E.) was:270.49±112.35 and 91.72±33.17, for Groups 1 and 2, respectively. Meanbody weights (±SE) at termination were: 25.36±1.07 and 24.25±1.68 forGroups 1 and 2, respectively. At the end of the study, the mean tumorgrowth inhibition in the SBT-100 treated group was 85.8% (p=0.006). FIG.7 illustrates the mean tumor volume. The tumor doubling times were 25.78days versus 111.6 days for Group 1 and Group 2, respectively. The %treatment/control for Group 2 was 13.35 (tumor inhibition).

TABLE 28 Mean Body Weights for Mice in MDA-MB-231 Phase Dosing RecoveryGroup Animal ID 8/28 9/1 9/4 9/8 9/11 9/15 9/18 Control 001 23.40 24.8024.60 25.00 24.70 23.30 25.10 Control 002 22.40 22.50 22.60 22.70 22.8020.60 23.10 Control 003 23.70 24.80 25.20 24.80 24.30 23.30 25.20Control 004 23.70 24.70 25.10 25.30 24.90 22.90 25.40 Mean 23.30 24.2024.38 24.45 24.18 22.53 24.70 Median 23.55 24.75 24.85 24.90 24.50 23.1025.15 SD 0.62 1.13 1.21 1.18 0.95 1.30 1.07 % Change 0.00 3.82 4.56 4.893.73 −3.38 5.97 SBT-100 005 21.70 21.70 21.70 22.40 22.60 21.40 22.20SBT-100 006 25.00 24.30 24.30 24.70 25.30 24.40 25.00 SBT-100 007 22.6023.00 23.10 23.10 23.80 22.80 23.70 SBT-100 008 26.50 25.30 25.50 26.1025.80 25.60 26.10 Mean 23.7 23.575 23.65 24.075 24.375 23.55 24.25Median 23.8 23.65 23.7 23.9 24.55 23.6 24.35 SD 1.84 1.56 1.63 1.66 1.461.84 1.68 % Change 0.00 −0.45 −0.15 1.65 2.96 −0.63 2.38

TABLE 29 Tumor Volumes for MDA-MB-231 Phase Animal Pre-Dosing DosingRecovery Group ID 8/21 8/24 8/27 8/28 9/1 9/4 9/8 9/11 9/15 9/18 Control001 51.00 55.80 80.94 76.35 83.66 94.11 110.78 129.99 162.81 184.15Control 002 75.19 77.22 121.13 120.73 145.12 179.21 203.16 234.05 308.70428.44 Control 003 57.04 57.81 75.32 81.06 93.25 114.27 181.87 242.88295.93 408.67 Control 004 42.92 51.67 106.54 92.23 116.96 142.60 191.58213.48 296.91 303.19 Mean 56.54 60.63 95.98 92.59 109.75 132.55 171.84205.10 263.59 331.11 Median 54.02 56.80 93.74 86.64 105.10 128.44 186.72223.76 291.42 355.93 SD 13.71 11.36 21.58 19.91 27.42 36.92 41.83 51.5767.78 112.35 SBT-100 005 72.25 64.45 80.02 74.07 56.81 49.44 68.70 73.0493.32 116.07 SBT-100 006 61.60 63.06 80.67 79.60 71.92 67.08 87.64115.80 116.97 120.44 SBT-100 007 37.41 35.15 91.93 91.85 50.02 50.3246.10 63.85 66.57 80.57 SBT-100 008 43.80 56.96 86.22 79.94 69.23 60.1954.10 82.57 79.47 49.78 Mean 53.74 54.91 84.71 81.37 59.49 56.76 64.1183.81 89.08 91.72 Median 52.65 60.02 83.45 79.77 58.02 55.25 61.40 77.8186.40 98.32 SD 18.00 13.57 6.56 7.49 9.16 0.43 18.21 22.65 21.56 33.17 %T/C 0.0 32.3 84.0 81.6 13.6 5.9 9.2 20.7 17.6 13.4 p-value 0.800 0.5420.351 0.332 0.013 0.007 0.003 0.005 0.003 0.006

In the DU145 tumor xenograft model, animals were randomized on day 17post-inoculation with a mean (±SE) tumor size of: 111.87±20.53 and111.23±25.16 for Groups 1 and 2, respectively. Mean body weights (±SE)at randomization were: 29.10±1.94 and 30.68±1.56 for Groups 1 and 2,respectively. Table 30 summarizes the mean body weights (±SE) for entirestudy. At last day of dosing (Day 14), the mean tumor size (±SE) forcontrol group was 621.81±276.25 versus 364.14±51.64 for SBT-100 treatedmice. Table 31 summarizes the tumor volumes (±SE) for entire study. Atthe time of termination (day 28) mean tumor size (±S.E.) was:819.42±351.88 and 601.83±131.51, for Groups 1 and 2, respectively. Meanbody weights (±SE) at termination were: 29.20±2.33 and 29.60±1.04 forGroups 1 and 2, respectively. At the end of the study, the mean tumorgrowth inhibition in the SBT-100 treated group was 26.6% (p=0.29). FIG.8 illustrates the mean tumor volume. The tumor doubling times were 14.57days versus 18.19 days for Group 1 and Group 2, respectively. The %treatment/control for Group 2 was 74.8.

TABLE 30 Mean Body Weights for Mice in DU145 Phase Dosing Recovery GroupAnimal ID 9/4 9/8 9/11 9/15 9/18 9/22 9/25 Control 001 29.60 28.10 29.3028.40 28.30 29.00 29.90 Control 002 29.70 30.10 31.20 30.10 30.40 29.9030.00 Control 003 30.80 30.10 31.00 31.70 31.20 31.10 31.10 Control 00426.30 25.70 26.60 25.20 26.10 26.20 25.80 Mean 29.10 29.50 29.53 28.8529.00 29.05 29.20 Median 29.65 29.10 30.15 29.25 29.35 29.45 29.95 SD1.94 2.09 2.13 2.78 2.29 2.09 2.33 % Change 0.00 −2.07 1.46 −0.99 −0.37−0.19 0.27 SBT-100 005 30.90 30.20 27.90 29.80 29.90 30.50 30.10 SBT-100006 28.40 26.20 27.30 26.90 27.50 29.10 28.50 SBT-100 007 31.70 31.2031.50 30.40 30.70 31.20 30.80 SBT-100 008 31.70 29.70 30.20 28.20 28.1028.80 29.00 Mean 30.68 29.33 29.23 28.83 29.05 29.90 29.60 Median 31.3029.95 29.05 29.00 29.00 29.80 29.55 SD 1.56 2.17 1.97 1.58 1.50 1.141.04 % Change 0.00 −4.47 −4.74 −6.00 −5.23 −2.39 −3.40

TABLE 31 Tumor Volumes for DU145 Phase Animal Pre-Dosing Dosing RecoveryGroup ID 8/24 8/27 8/31 9/3 9/4 9/8 9/11 9/15 9/18 9/22 9/25 Control 00138.18 41.27 38.41 82.60 83.22 121.16 203.79 310.41 409.15 430.31 450.69Control 002 45.05 35.99 64.98 96.50 103.83 135.42 225.62 327.76 478.14534.48 599.97 Control 003 49.65 22.37 88.76 127.98 141.49 213.24 334.15930.74 1,023.13 1,084.00 1,188.93 Control 004 17.06 36.44 86.73 131.20138.33 227.23 289.78 338.79 576.83 926.90 1,027.90 Mean 38.98 34.0264.47 111.87 119.22 174.26 275.84 476.93 621.81 743.95 819.42 Median42.11 36.22 65.36 111.74 121.98 174.33 257.70 333.27 527.49 730.59813.93 SD 13.67 8.12 20.58 20.53 24.32 53.70 80.91 302.77 276.25 311.67351.98 SBT-100 005 33.80 23.32 35.67 86.02 89.21 151.52 145.67 386.92325.88 474.31 496.83 SBT-100 006 59.44 41.00 64.21 98.56 121.39 148.44208.10 357.62 391.02 518.25 588.67 SBT-100 007 42.20 35.11 77.90 144.66145.78 115.05 106.70 248.12 316.24 454.78 528.83 SBT-100 008 63.37 50.1871.23 116.28 118.70 134.16 147.52 320.22 423.45 604.72 793.96 Mean 51.2337.40 59.75 111.23 118.77 137.39 151.50 328.22 364.14 513.01 601.83Median 50.87 38.06 82.72 107.42 120.05 141.30 146.80 338.92 368.43496.20 558.76 SD 15.13 11.25 18.90 25.18 23.17 16.78 40.98 59.97 51.6466.65 131.51 % T/C 0.0 −26.8 29.1 78.8 81.7 65.4 42.1 70.6 56.9 69.974.8 p-value 0.226 0.643 0.747 0.970 0.990 0.238 0.034 0.372 0.115 0.1970.291

In the PANC-1 tumor xenograft model, animals were randomized on day 22post-inoculation with a mean (±SE) tumor size of: 78.74±40.21 and93.84±36.31 for Groups 1 and 2, respectively. Mean body weights (±SE) atrandomization were: 22.50±1.47 and 24.23±1.63 for Groups 1 and 2,respectively. Table 32 summarizes the mean body weights (±SE) for entirestudy. At last day of dosing (Day 14), the mean tumor size (±SE) forcontrol group was 204.95±178.90 versus 159.03±28.01 for SBT-100 treatedmice. Table 33 summarizes the tumor volumes (±SE) for entire study. Atthe time of termination (day 28) mean tumor size (±S.E.) was:284.77±288.88 and 203.02±30.34, for groups 1 and 2, respectively. Meanbody weights (±SE) at termination were: 27.38±1.07 and 26.23±1.19 forGroups 1 and 2, respectively. At the end of the study, the mean tumorgrowth inhibition in the SBT-100 treated group was 41.78% (p=0.35). FIG.9 illustrates the mean tumor volume. The tumor doubling times were 18.51days versus 35.70 days for Group 1 and Group 2, respectively. The %treatment/control for Group 2 was 52.79.

TABLE 32 Mean Body Weights for Mice in PANC-1 Phase Dosing RecoveryGroup Animal ID 9/9 9/11 9/15 9/18 9/22 9/25 9/29 Control 001 26.5026.60 25.60 27.10 25.70 26.10 27.20 Control 002 24.30 24.60 23.90 25.1024.40 25.00 25.60 Control 003 27.60 26.50 26.30 26.20 26.10 27.60 28.20Control 004 25.10 25.30 24.20 25.10 24.70 25.90 26.90 Mean 22.50 22.8023.04 24.30 24.58 25.90 27.38 Median 25.60 25.90 25.00 25.65 25.20 26.0027.05 SD 1.47 0.97 1.18 0.97 0.81 1.03 1.07 % Change 0.00 −0.39 −3.150.12 −2.41 1.05 4.33 SBT-100 005 22.60 22.80 21.40 22.60 22.60 22.9024.60 SBT-100 006 26.00 25.10 24.90 25.70 25.10 25.40 27.10 SBT-100 00723.10 22.30 22.40 22.70 23.10 23.50 25.70 SBT-100 008 26.20 26.00 25.2025.40 26.20 25.40 27.30 Mean 24.23 23.80 23.48 24.08 24.25 24.30 26.23Median 24.15 23.90 23.65 24.05 24.10 24.45 26.40 SD 1.63 1.46 1.87 1.711.69 1.29 1.19 % Change 0.00 −1.71 −3.14 −0.63 0.13 0.39 8.39

TABLE 33 Tumor Volumes for PANC-1 Phase Animal Pre-Dosing DosingRecovery Group ID 8/31 9/3 9/6 9/9 9/11 9/15 9/18 9/22 9/25 9/29 Control001 54.91 56.79 94.23 94.37 94.69 123.90 136.77 206.74 220.31 223.91Control 002 46.38 75.43 81.99 81.62 88.44 130.01 161.06 140.52 146.62202.22 Control 003 0.00 27.50 57.30 59.60 99.77 107.00 142.23 140.55168.68 187.27 Control 004 0.00 0.00 152.17 159.98 227.02 380.54 502.66514.93 574.44 781.45 Mean 20.26 32.54 78.74 80.91 104.18 151.29 189.83204.95 226.81 284.77 Median 23.19 42.15 88.11 87.99 97.23 126.95 146.64173.65 194.50 213.06 SD 29.45 33.13 40.21 43.18 66.52 130.48 179.63178.90 200.56 288.88 SBT-100 005 39.60 64.75 76.44 78.07 57.54 93.17112.98 140.09 173.92 245.84 SBT-100 006 40.31 37.27 68.57 73.13 76.46113.30 130.49 192.56 205.65 189.42 SBT-100 007 85.71 91.27 147.61 149.02123.96 116.01 157.50 171.29 176.68 200.97 SBT-100 008 48.72 65.19 82.7383.18 86.90 102.48 106.65 132.19 136.93 175.84 Mean 53.58 62.12 83.8495.85 86.21 106.24 126.65 158.03 173.02 203.02 Median 44.51 59.97 79.5980.62 81.68 107.89 121.73 155.69 174.80 195.19 SD 21.82 22.62 36.3135.69 27.94 10.49 23.06 28.01 28.10 30.34 % T/C 0.0 0.0 42.6 44.2 27.434.4 38.0 49.7 51.0 52.6 p-value 0.174 0.310 0.927 0.917 0.296 0.2720.286 0.350 0.343 0.355

Example 12 Efficacy of Anti-STAT3 Bacterial VHH13 (SEQ ID NO:3) sdAb inthe ER+/PR+ (MCF-7) Human Breast Tumor Xenograft Model

This Example demonstrates the efficacy of anti-STAT3 bacterial VHH13(SEQ ID NO:3) sdAb in the MCF-7 human breast tumor xenograft model innude mice.

Female athymic nude mice (Crl:NU(Ncr)-Foxn1^(nu), Charles River) weretwelve weeks old with a body weight (BW) range of 23.0 to 30.1 g on Day1 of the study. The animals were fed and housed as described above.

MCF-7 human breast carcinoma cells were obtained and cultured asdescribed above, and used for the mouse xenograph. Three days prior totumor cell implantation, estrogen pellets (0.36 mg estradiol, 60-dayrelease, Innovative Research of America, Sarasota, Fla.) were implantedsubcutaneously between the scapulae of each test animal using asterilized trocar.

The tumor cells used for implantation were harvested during log phasegrowth and resuspended in phosphate buffered saline (PBS) at aconcentration of 1×10⁸ cells/mL. On the day of implantation, each testmouse received 1×10⁷ MCF-7 cells (0.1 mL cell suspension) implantedsubcutaneously in the right flank and tumor growth was monitored as theaverage size approached the target range of 100-150 mm³. Twenty-one dayslater, designated as Day 1 of the study, the animals were sorted intotwo groups each consisting of four mice with individual tumor volumesranging from 108 to 144 mm³ and group mean tumor volumes from 117 to 123mm³.

Anti-STAT3 bacterial VHH13 (SEQ ID NO:3) sdAb was provided as apre-formulated ready to dose solution at a concentration 0.41867 mg/mLin 1 mL aliquots and were stored at −20° C. until needed. The 0.41867mg/mL solution provided 1 mg/kg dosage in a dosing volume of 23.88mL/kg. On each day of treatment, only needed vials of anti-STAT3bacterial VHH13 (SEQ ID NO:3) sdAb were thawed to room temperature. Allleftover dosing suspensions were retained at 4° C. as needed for thenext dose.

Two groups of athymic nude mice were dosed according to the protocolshown in Table 34. All vehicle (control) and anti-STAT3 bacterial VHH13(SEQ ID NO:3) sdAb doses were administered intraperitoneally (i.p.)three times daily, six hours apart for fourteen days, with two dosesdelivered on Day 1 and one dose delivered on the morning of Day 15(tid×14, first day 2 doses). The dosing volume for vehicle andanti-STAT3 bacterial VHH13 (SEQ ID NO:3) sdAb was 0.478 mL per 20 gramsof body weight (23.88 mL/kg) and was scaled to the body weight of eachindividual animal. Group 1 received the vehicle and served as thebenchmark group for tumor engraftment and progression, as well as thecontrol. Group 2 was given anti-STAT3 bacterial VHH13 (SEQ ID NO:3) sdAbat 1 mg/kg.

TABLE 34 Protocol Design for the Study Treatment Regimen Group n Agentmg/kg Route Schedule 1 4 vehicle — ip tid × 14 first Day 2 doses 2 4VHH13 1 ip tid × 14 first Day 2 doses

Tumors were measured twice weekly, and each animal was euthanized whenits neoplasm reached the predetermined endpoint volume (1000 mm³) or atthe end of the study, day 39, whichever came first. When a tumor reachedthe endpoint volume, the animal was documented as euthanized for tumorprogression (TP), with the date of euthanasia. The time to endpoint(TTE) for each mouse was calculated by the following equation:

${TTE} = \frac{{\log_{10}\left( {{endpoint}\mspace{14mu} {volume}} \right)} - b}{m}$

where TTE is expressed in days, endpoint volume is expressed in mm³, bis the intercept, and m is the slope of the line obtained by linearregression of a log-transformed tumor growth data set. The data setconsists of the first observation that exceeded the endpoint volume usedin analysis and the three consecutive observations that immediatelypreceded the attainment of this endpoint volume. The calculated TTE isusually less than the TP date, the day on which the animal waseuthanized for tumor size. Animals that did not reach the endpointvolume were assigned a TTE value equal to the last day of the study(D39). Any animal classified as having died from treatment-related (TR)causes was to be assigned a TTE value equal to the day of death. Anyanimal classified as having died from non-treatment-related (NTR) causeswas to be excluded from TTE calculations.

Treatment efficacy was determined from tumor growth delay (TGD), whichis defined as the increase in the median TTE, in days, for a treatmentgroup compared to the control group:

TGD=T−C

The percent increase in the median TTE, relative to the control group,is

where:

${\% \; {TGD}} = {\frac{T - C}{C} \times 100}$

T=median TTE for a treatment group, and

C=median TTE for the designated control group.

Treatment efficacy in each group may be indicated by the median tumorvolume, MTV(n), which was defined as the median tumor volume on the lastday of the study (D39) in the number of animals remaining (n) whosetumors had not attained the endpoint volume.

Treatment efficacy may also be determined from the incidence andmagnitude of regression responses observed during the study. Treatmentmay cause partial regression (PR) or complete regression (CR) of thetumor in an animal. In a PR response, the tumor volume was 50% or lessof its D1 volume for three consecutive measurements during the course ofthe study, and equal to or greater than 13.5 mm³ for one or more ofthese three measurements. In a CR response, the tumor volume was lessthan 13.5 mm³ for three consecutive measurements during the course ofthe study. Any animal with a CR response at the end of the study wasadditionally classified as a tumor-free survivor (TFS).

Animals were weighed daily for the first five days, then twice weeklyfor the remainder of the study. The mice were observed frequently forhealth and overt signs of any adverse treatment related TR side effects,and noteworthy clinical observations were recorded. Individual bodyweight loss was monitored per protocol, and any animal with weight lossexceeding 30% for one measurement, or exceeding 25% for threemeasurements, was to be euthanized for health as a TR death. If groupmean body weight recovered, dosing may resume in that group, but at alower dose or less frequent dosing schedule. Acceptable toxicity wasdefined as a group mean BW loss of less than 20% during the study andnot more than one TR death among ten treated animals, or 10%. Any dosingregimen resulting in greater toxicity is considered above the maximumtolerated dose (MTD). A death was to be classified as TR if it wasattributable to treatment side effects as evidenced by clinical signsand/or necropsy, or may also be classified as TR if due to unknowncauses during the dosing period or within 14 days of the last dose. Adeath was classified as NTR if there was evidence that the death wasrelated to the tumor model, rather than treatment-related. NTR deathsare further categorized as NTRa (due to accident or human error), NTRm(due to necropsy-confirmed tumor dissemination by invasion ormetastasis), and NTRu (due to unknown causes).

Prism 6.07 (GraphPad) for Windows was employed for graphical analyses.Statistics were not employed due to small sample size.

A scatter plot was constructed to show TTE values for individual mice,by group; this plot shows NTR deaths, which were excluded from all otherfigures. Individual animal, group median and mean tumor volumes wereplotted as functions of time. When an animal exited the study because oftumor size or TR death, its final recorded tumor volume was includedwith the data used to calculate the median volume at subsequent timepoints. A Kaplan-Meier plot was constructed to show the percentage ofanimals in each group remaining on study versus time. Tumor growthcurves were truncated after two TR deaths occurred in the same group.Group mean BW changes over the course of the study were graphed aspercent change, ±SEM, from Day 1. Tumor growth and BW change curves weretruncated after more than half the assessable mice in a group exited thestudy. FIG. 10 illustrates the mean tumor volume in the study.

Table 35 provides the mean BW losses, TR and NTR deaths for the mice.Clinical signs were recorded when observed, as shown in Tables 36-38. NoTR deaths occurred during the study. Bodyweight losses were variable,severe for one animal in each group, and resulted from estrogen effects.Clinical observations including weight loss, enlarged uterine horns, andbladder crystals were present in both groups and were also attributableto estrogen effects. Estrogen toxicity resulted in two non-treatmentrelated deaths in each group. The treatment evaluated in the study wasacceptably tolerated.

TABLE 35 Response Summary Treatment Regimen Median MTV (n) RegressionsMean BW Deaths Group n Agent mg/kg Route Schedule TTE T-C % TGD D39 PRCR TFS Nadir TR NTR 1 2 vehicle — ip tid × 14 first 23.2 — — — 0 0 0−15.6% 0 2 Day 2 doses Day 25 2 2 VHH13 1 ip tid × 14 first 32.9 9.7 42— 0 0 0 −21.9% 0 2 Day 2 doses Day 32

TABLE 36 Body Weight Body Weight Date Jul. 27, 2015 Jul. 28, 2015 Jul.29, 2015 Jul. 30, 2015 Jul. 31, 2015 Aug. 3, 2015 Aug. 6, 2015 Aug. 10,2015 Day of Study 1 2 3 4 5 8 11 15 A# Wt (g) Wt (g) Wt (g) Wt (g) Wt(g) Wt (g) Wt (g) Wt (g) Group 1: vehicle (ip, tid × 14 first Day 2doses) 1 27.50 28.60 28.10 29.40 NTRa on Aug. 1, 2015 2 26.30 27.3027.40 29.30 27.40 26.80 26.40 26.50 3 30.10 31.00 30.50 27.30 31.1029.50 28.20 26.00 4 23.00 24.20 24.40 30.00 25.00 NTRu on Aug. 3, 2015Mean 26.7 27.8 27.6 27.9 28.2 28.2 27.3 26.3 STDEV 2.9 2.8 2.5 2.2 2.61.9 1.3 0.4 n 4 4 4 4 4 2 2 2 Group 2: VHH13 (1 mg/kg, ip, tid × 14first Day 2 doses) 1 28.90 29.30 28.50 29.60 28.80 28.70 27.80 28.30 225.30 27.00 26.40 26.40 26.30 25.90 25.80 26.00 3 27.20 25.40 23.90 NTRuon Jul. 30, 2015 4 27.60 27.50 27.10 27.30 27.20 26.90 26.10 NTRu onAug. 8, 2015 Mean 27.3 27.3 26.5 27.8 27.4 27.2 26.6 27.2 STDEV 1.5 1.61.9 1.7 1.3 1.4 1.1 1.6 n 4 4 4 3 3 3 3 2 Date Aug. 13, 2015 Aug. 17,2015 Aug. 20, 2015 Aug. 24, 2015 Aug. 27, 2015 Aug. 31, 2015 Sep. 3,2015 Day of Study 18 22 25 29 32 36 39 A# Wt (g) Wt (g) Wt (g) Wt (g) Wt(g) Wt (g) Wt (g) Group 1: vehicle (ip, tid × 14 first Day 2 doses) 1NTRa on Aug. 1, 2015 2 26.60 27.20 TP on Aug. 17, 2015 3 22.50 23.6023.80 TP on Aug. 20, 2015 4 NTRu on Aug. 3, 2015 Mean 24.6 25.4 23.8STDEV 2.9 2.5 n 2 2 1 Group 2: VHH13 (1 mg/kg, ip, tid × 14 first Day 2doses) 1 28.20 28.80 29.00 28.90 TP on Aug. 24, 2015 2 24.50 21.90 20.1021.10 21.20 23.80 24.20 3 NTRu on Jul. 30, 2015 4 NTRu on Aug. 8, 2015Mean 26.4 25.4 24.6 25 21.2 23.8 24.2 STDEV 2.6 4.9 6.3 5.5 n 2 2 2 2 11 1

TABLE 37 Tumor Measurement Caliper Measurement Date Jul. 27, 2015 Jul.30, 2015 Aug. 3, 2015 Aug. 6, 2015 Aug. 10, 2015 Aug. 13, 2015 Day ofStudy 1 4 8 11 15 18 A# W (mm) L (mm) W (mm) L (mm) W (mm) L (mm) W (mm)L (mm) W (mm) L (mm) W (mm) L (mm) Group 1: vehicle (ip, tid × 14 firstDay 2 doses) 1 6 6 7 8 NTRa on Aug. 1, 2015 2 5 9 7 9 8 12 9 13 10 13 1013 3 6 7 7 10 9 10 10 12 11 12 11 12 4 6 8 7 11 NTRu on Aug. 3, 2015Group 2: VHH13 (1 mg/kg, ip, tid × 14 first Day 2 doses) 1 6 6 7 8 8 109 10  9 10  9 10 2 6 6 6 7 7 8 7 8 8 9 8 9 3 6 7 4 6 7 6 8 7 10 8 10Date Aug. 17, 2015 Aug. 20, 2015 Aug. 24, 2015 Aug. 27, 2015 Aug. 31,2015 Sep. 3, 2015 Day of Study 22 25 29 32 36 39 A# W (mm) L (mm) W (mm)L (mm) W (mm) L (mm) W (mm) L (mm) W (mm) L (mm) W (mm) L (mm) Group 1:vehicle (ip, tid × 14 first Day 2 doses) 1 NTRa on Aug. 1, 2015 2 12 15TP on Aug. 17, 2015 3 11 13 12 14 TP on Aug. 20, 2015 4 NTRu on Aug. 3,2015 Group 2: VHH13 (1 mg/kg, ip, tid × 14 first Day 2 doses) 1 10 11 1212 13 13 TP on Aug. 24, 2015 2 9 10 9 10 10 10 10 10 12 12 13 13 3 NTRuon Jul. 30, 2015 4 NTRu on Aug. 8, 2015

TABLE 38 Tumor Volume Tumor Volume Group 1: vehicle (ip, tid × 14 firstDay 2 doses) Date Jul. 27, 2015 Jul. 30, 2015 Aug. 3, 2015 Aug. 6, 2015Aug. 10, 2015 Aug. 13, 2015 Aug. 17, 2015 Day of Study 14 8 11 15 18 22A# TV (mm³) TV (mm³) TV (mm³) TV (mm³) TV (mm³) TV (mm³) TV (mm³) 1 196NTRa on Aug. 1, 2015 2 108 221 384 527 650 650 1080 3 113 245 405 600726 726 787 4 126 270 NTRu on Aug. 3, 2015 Mean 122.6 232.8 394.5 563.3688 688 933.3 SEM 8.1 15.8 10.5 36.8 38 38 146.8 n 4 4 222221 Date Aug.20, 2015 Aug. 24, 2015 Aug. 27, 2015 Aug. 31, 2015 Sep. 3, 2015 Day ofStudy 25 29 32 36 39 A# TV (mm³) TV (mm³) TV (mm³) TV (mm³) TV (mm³) 1NTRa on Aug. 1, 2015 2 TP on Aug. 17, 2015 3 1008 TP on Aug. 20, 2015 4NTRu on Aug. 3, 2015 Mean 1008 SEM n Group 2: VHH13 (1 mg/kg, ip, tid ×14 first Day 2 doses) Date Jul. 27, 2015 Jul. 30, 2015 Aug. 3, 2015 Aug.6, 2015 Aug. 10, 2015 Aug. 13, 2015 Aug. 17, 2015 Day of Study 14 8 1115 18 22 A# TV (mm³) TV (mm³) TV (mm³) TV (mm³) TV (mm³) TV (mm³) 1 108196 320 405 405 405 550 2 108 126 196 196 288 288 405 3 126 NTRu on Jul.30, 2015 4 126 144 245 320 NTRu on Aug. 8, 2015 Mean 117 155.3 253.7 307346.5 346.5 477.5 SEM 5.2 21 36.1 60.7 58.5 58.5 72.5 n 4 3 3 Date Aug.20, 2015 Aug. 24, 2015 Aug. 27, 2015 Aug. 31, 2015 Sep. 3, 2015 Day ofStudy 25 29 32 36 39 A# TV (mm³) TV (mm³) TV (mm³) TV (mm³) TV (mm³) 1864 1099 TP on Aug. 24, 2015 2 405 500 500 864 1099 3 NTRu on Jul. 30,2015 4 NTRu on Aug. 8, 2015 Mean 634.5 799.3 500 864 1098.5 SEM 229.5299.3 n 322222111

Because two out of the four mice in the control group and also in thetreatment group died of estrogen toxicity, no statistical conclusioncould be determined. With the data available, the median tumor growthand mean tumor volume were reduced in the treatment group when comparedto the control group. This difference was present during the 14 days oftreatment but also to day 25 of the study. It took the control group 25days to reach a tumor volume of 1000 mm³, whereas the treatment grouptook 36 days to reach a tumor volume of 1000 mm³. This suggests thatanti-STAT3 bacterial VHH13 (SEQ ID NO:3) sdAb slows the growth of MCF-7tumor in vivo. Throughout the study both the control group and thetreatment group maintained similar weights. This suggests that theanti-STAT3 bacterial VHH13 (SEQ ID NO:3) sdAb did not cause toxicitywith respect to weight loss.

Example 13 Treatment of Human HER2+ (BT474) Breast Cancer withAnti-STAT3 Bacterial VHH13 (SEQ ID NO:3) sdAb in Xenograft Mice

In this Example, the efficacy of anti-STAT3 bacterial VHH13 (SEQ IDNO:3) sdAb was determined in the BT474 human breast tumor xenograft inCB.17 SCID mice.

Two groups of 8-12 week old CB.17 SCID mice containing xenographs of 1mm³ BT474 tumor fragments in their flank were treated according to theprotocol shown in Table 39 when the tumors reached an average size of100-150 mm³. All vehicle (PBS control) and anti-STAT3 bacterial VHH13(SEQ ID NO:3) sdAb (shown in Table 39 as SB-01) doses were administeredintraperitoneally (i.p.) three times daily, six hours apart for fourteendays, with two doses delivered on Day 1 (tid×14, first day 2 doses). Thedosing volume for vehicle and anti-STAT3 bacterial VHH13 (SEQ ID NO:3)sdAb was 0.478 mL per 20 grams of body weight (23.88 mL/kg) and wasscaled to the body weight of each individual animal. Group 1 receivedthe vehicle and served as the benchmark group for tumor engraftment andprogression, as well as the control. Group 2 was given anti-STAT3bacterial VHH13 (SEQ ID NO:3) sdAb at 1 mg/kg.

TABLE 39 Study protocol Regimen 1 Gr. N Agent Vehicle mg/kg RouteSchedule 1^(#) 4 vehicle — ip tid × 14 first day 2 doses 2 4 SB-01 1 iptid × 14 first day 2 doses

During the first 14 days of the study, the treatment group receivedanti-STAT3 B VHH13 and the control group only received the vehicle. Asshown in Table 40, during this time, the treatment group maintained andgained weight throughout the study while the control group had lowerweights throughout the study. This suggests that the treatment group didnot experience toxicity from anti-STAT3 bacterial VHH13 (SEQ ID NO:3)sdAb with respect to weight loss. Both groups mean tumor volume andmedian tumor volume were similar, and exactly the same on day 15 of thestudy. On day 59 of the study, both groups reached a tumor volume of 700cubic mm³. This suggests that the anti-STAT3 bacterial VHH13 (SEQ IDNO:3) sdAb did not reduce the growth of BT474 tumors in vivo whencompared to the control group. FIG. 11 illustrates the group mean tumorvolume.

TABLE 40 BT474 Response Summary MTV Treatment Regimen 1 Median % Stat(n), BW NTR Group n Agent Vehicle mg/kg Route Schedule TTE T-C TGD SignDay 60 PR CR TFS Nadir TR NTRm NTR 1^(#) 4 vehicle — ip tid × 14 49.2 —— 288 (2) 0 0 0 −9.1% 0 0 0 first day (3) 2 doses 2 4 SB-01 1 ip tid ×14 60.0 10.8 22 550 (3) 0 0 0 — 0 0 0 first day 2 doses ^(#)ControlGroup

Example 14 Production of Mouse Monoclonal Antibody Directed AgainstAnti-STAT3 Bacterial VHH13 (SEQ ID NO:3) sdAb

In this Example, mouse monoclonal antibodies were generated towards thesdAb of the invention. The animals used were BALB/c female mice, 8-10week. A water-soluble adjuvant was used (CBL). The HAT and the HT usedwere from Sigma-Aldrich.

Anti-STAT3 bacterial VHH13 (SEQ ID NO:3) sdAb was used to immunize threemice and make hybridoma cell lines. The mice were immunized three timeseach with water-soluble adjuvant. In one mouse, the serum titer reached1/51200. The mouse was sacrificed and hybridoma cell lines were made byfusing spleen cells with myeloma cell line Sp2/0.

The fused cells were seeded into 96 well plates by limited dilution. Thefused cells were cultured in the presence of HAT, and 651 single cloneswere tested. Of the 651 single clones, 27 positive clones wereidentified that specifically bound to anti-STAT3 bacterial VHH13 (SEQ IDNO:3) sdAb antigen.

Example 15 Cytotoxicity of KRAS (G12D) Single Domain Antibodies onPANC-1 Human Pancreatic Cancer Cells

This Example demonstrates the anti-proliferative effects of theanti-KRAS (G12D) (SEQ ID NO:2) sdAb using the human pancreatic cancercell line PANC-1. For the experiments, the PANC-1 cells were grown untilthey reached a confluency of 90%. At that time, proliferation studieswere carried out using the MTT assay as described above.

The anti-proliferative properties of anti-KRAS (G12D) (SEQ ID NO:2) sdABon PANC-1 cells three days post treatment are shown in Table 41. PANC-1cells treated with the anti-KRAS (G12D) (SEQ ID NO:2) sdAb showed anaverage growth inhibition of 19.9 and 37.7 at 50.0 and 100 μg/ml,respectively.

TABLE 41 Anti-proliferative Actions of Anti-KRAS (G12D) (SEQ ID NO: 2)sdAb on PANC-1 Cancer Cells Mean Abs ± SE % Inhibition control 0.281 ±0.017  50 μg/ml 0.225 ± 0.006 19.9 100 μg/ml 0.175 ± 0.016 37.7

Thus, the anti-KRAS (G12D) (SEQ ID NO:2) sdAb showed dose-dependentgrowth inhibition in the PANC-1 human pancreatic cancer cells.

Example 16 In Vitro Growth Inhibition by TNF-Alpha sdAB

This Example demonstrates the method development to determine TNF-alphaconcentration and evaluation of the inhibition of TNF-alpha function.The concentration of TNF-alpha required to show measurable modulation ofactivity in the U937 human lung lymphoblast cell line was evaluated byquantitation of the ATP present, which signals the presence ofmetabolically active cells using Promega's Cell Titer-GJo® LuminescentCell Viability assay.

The U937 cells were seeded in a clear polystyrene 96-well microcultureplate (Corning® Costar® 96-well flat bottom plate, Cat. #3997) in atotal volume of 90 μL/well. After 24 hours of incubation in a humidifiedincubator at 37° C. with 5% CO₂ and 95% air, 5 μL of 20×, seriallydiluted TNF-alpha in growth medium was added to each well in duplicate(10 pt dose response, highest concentration 20 ng/mL). Additionally, 5μL of 20×, diluted staurosporine in growth medium was added to each wellin duplicate (concentration 1 nM).

After 24 hours of culture in the presence of test agents, theconcentration of compound required to show measurable modulation ofTNF-alpha activity in the U937 cell line as evaluated by quantitation ofthe ATP present. Percent cell growth was calculated relative tountreated control wells. All tests were performed in duplicate at eachconcentration level.

The EC₅₀ value for the test agents was estimated using Prism 6.05 bycurve-fitting the data using the following four parameter-logisticequation:

$Y = {\frac{{Top} - {Bottom}}{1 + \left( \frac{X}{\left. {IC}_{50} \right)} \right)^{n}} + {Bottom}}$

where Top is the maximal % of control absorbance, Bottom is the minimal% of control absorbance at the highest agent concentration, Y is the %of control absorbance, X is the agent concentration, IC₅₀ is theconcentration of agent that inhibits cell growth by 50% compared to thecontrol cells, and n is the slope of the curve.

FIGS. 12 and 13 demonstrate that TNF-alpha is cytotoxic to the U937cells. The IC₅₀ for TNF-alpha against U937 is 95.10 pg/ml. The TNF-alphacurve shows a dose titration killing effect.

FIG. 14 demonstrates that TNF-alpha cytotoxicity against U937 isinhibited by the three different anti-TNF-alpha VHHs. Whenanti-TNF-alpha VHH62 (SEQ ID NO:47) sdAb, anti-TNF-alpha VHH 66 (SEQ IDNO:45) sdAb, and anti-TNF-alpha VHH69 (SEQ ID NO:46) sdAb were incubatedwith a constant dose of TNF-alpha, at EC₅₀, all three anti-TNF-alphaVHHs inhibit killing of U937 by TNF-alpha. The IC₅₀ of anti-TNF-alphaVHH62 (SEQ ID NO:47) sdAb was approximately 713.6 ug/ml. The IC₅₀ ofanti-TNF-alpha VHH69 (SEQ ID NO:46) sdAb was greater than 208.055 ug/ml.The IC₅₀ of anti-TNF-alpha VHH66 (SEQ ID NO:45) sdAb could not bedetermined because it completely inhibited the cytotoxicity of TNF-alphafrom concentrations of about 1×10² ug/ml to 1×10² ug/ml ofanti-TNF-alpha VHH66 (SEQ ID NO:45) sdAb. In this concentration range ofanti-TNF-alpha VHH66 (SEQ ID NO:45) sdAb, there is an increase in U937cell growth, and thus complete inhibition of TNF-alpha activity.

Although the present invention has been described in considerable detailwith reference to certain preferred embodiments, other embodiments arepossible. The steps disclosed for the present methods, for example, arenot intended to be limiting nor are they intended to indicate that eachstep is necessarily essential to the method, but instead are exemplarysteps only. Therefore, the scope of the appended claims should not belimited to the description of preferred embodiments contained in thisdisclosure. All references cited herein are incorporated by reference intheir entirety.

What is claimed is:
 1. An anti-STAT3 single domain antibody (sdAb). 2.The anti-STAT3 sdAb of claim 1, wherein the anti-STAT3 sdAb comprises anamino acid sequence as set forth in SEQ ID NO:3 or SEQ ID NO:4.
 3. Amethod of treating a disease, preventing development of a disease, orpreventing recurrence of a disease in a subject using the anti-STAT3sdAb according to claim 2, the method comprising administering aneffective amount of the anti-STAT3 sdAb to a subject in need thereof. 4.The method of claim 3, wherein the subject is a mammal.
 5. The method ofclaim 4, wherein the mammal is a human.
 6. The method of claim 3,wherein the anti-STAT3 sdAb is administered in combination with one ormore compounds.
 7. The method of claim 6, wherein the one or morecompounds is a transcriptional inhibitor.
 8. The method of claim 3,wherein administering an effective amount of the anti-STAT3 sdAb to asubject in need thereof comprises intravenous administration,intramuscular administration, oral administration, rectaladministration, enteral administration, parenteral administration,intraocular administration, subcutaneous administration, transdermaladministration, administered as eye drops, administered as nasal spray,administered by inhalation or nebulization, topical administration, andadministered as an implantable drug.
 9. An isolated polypeptide, theisolated polypeptide comprising an amino acid sequence as set forth inSEQ ID NO:3 or SEQ ID NO:4.
 10. An antibody directed toward thepolypeptide of claim
 9. 11. A method of measuring the levels of ananti-STAT3 sdAb in a sample from a subject, the method comprising thesteps of: a) generating a mouse monoclonal antibody directed against oneor more domains of a polypeptide comprising an amino acid sequence asset forth in SEQ ID NO:3 or SEQ ID NO:4; b) obtaining a sample from thesubject; c) performing a quantitative immunoassay with the mousemonoclonal antibody and the sample to determine the amount of sdAb in asubject; and d) quantifying the amount of sdAb in the subject.
 12. Themethod of claim 11 wherein the quantitative immunoassay comprises anenzyme-linked immunosorbent assay (ELISA), specific analyte labeling andrecapture assay (SALRA), liquid chromatography, mass spectrometry,fluorescence-activated cell sorting, or a combination thereof.